Recombinant multiple domain fusion protein mitogens and use thereof for inducing enhancement or repression of antigen-specific immunity.

ABSTRACT

The invention relates to cell stimulatory fusion proteins and DNA sequences, vectors comprising at least two agonists of TNF/TNFR super family, immunoglobulin super family, cytokine family proteins and optional antigen combination. Instructions for use of these proteins and DNA constructs as immune adjuvants and vaccines for treatment of various chronic diseases such as viral infection are also provided. Additionally, the use of these protein and DNA constructs as immune suppressant for treatment of various chronic diseases, such as autoimmunity and organ transplant rejection, is also illustrated.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of application Ser. No. 12/483,876, filed Jun. 12, 2009, which claims priority to U.S. Provisional Patent Application No. 61/073,010, filed Jun. 16, 2008.

REFERENCE CITED [REFERENCE BY]

U.S. Patent Documents 6,410,711 Jun. 25, 2002 Armitage, et al. 6,838,262 Oct. 13, 2000 Anderson, et al. 6,046,310 Apr. 4, 2000 Queen, et al 5,962,406 Oct. 5, 1999 Armitage, et al. 5,942,607 Aug. 24, 1999 Freeman, et al. 5,457,035 Jul. 23, 1993 Baum, et al.

Foreign Patent Documents WO/2007/103048 Feb. 28, 2007 WO/2007/022273 Aug. 15, 2006 WO/2006/050949 Nov. 10, 2005 WO/2003/063899 Jan. 28, 2003 WO/1997/033617 Nov. 3, 1997 WO/1995/013293 May 18, 1995

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FIELD OF THE INVENTION

The invention generally relates to methods and compositions, which promote or repress antigen-specific immunity. The described polypeptide and DNA constructs are used as either immune adjuvants or suppressants for treating various chronic diseases including cancer, infectious diseases, autoimmune diseases, allergies and inflammatory diseases.

BACKGROUND OF THE INVENTION

Affording to Current Understanding of Molecular Mechanisms of Diseases, Reagents that Modulate Immune Responses are in Great Demand.

The body's defense system against microbes and other chronic diseases is mediated by the two main components of the immune system: the innate immune system and adaptive immune system. Recent advances in the study of the molecular and cellular mechanism of various diseases indicated that both innate and adaptive immune systems are targeted for the prevention and cure of various types of diseases. Innate immunity involves mechanisms that recognize structures, which are characteristic to microbial pathogens but are not present on mammalian cells. Examples of such structures include bacterial liposaccharides (LPS), viral double stranded DNA, and unmethylated CpG DNA nucleotides. The effector cells of the innate immune response system comprise neutrophils, macrophages, and natural killer cells (NK cells). In adaptive immunity, the body's immunological defense systems are stimulated by exposure to infectious agents and these responses increase in magnitude and effectiveness with each successive exposure to that particular antigen. There are two types of adaptive immune responses: (i) humoral immunity, which involves the production of pathogen-specific antibodies by B lymphocytes (B cells), and (ii) cell-mediated immunity, which is regulated by T lymphocytes (T cells). Immune effector cells in the innate and adaptive phases of immune responses can be directly or indirectly involved in the cause of some diseases, and are thus potentially important targets for therapeutics against these diseases. Recently, there has been an increasing number of vaccine strategies used against a variety of disease conditions. Infectious diseases caused by viruses, bacteria and parasites [Targett and Greenwood Malar J. 7 Suppl 1:S10 (2008); Okwor and Uzonna Hum Vaccin. May 12; 5 (2009)] are areas of on-going research using vaccines. Unexpectedly, vaccine strategies may also be effective against diseases such as Alzheimer's and prion diseases, in which the scavenger functions of immune response cells against pathogenic metabolic deposits is ineffective [Wisniewski and Konietzko, Lancet Neurol. 7:805 (2008); Sakaguchi, Protein Pept Lett., 16:260 (2009)]. Autoimmune diseases are also an area in which immune responses against the host self-components could potentially be inhibited at pathogenic level by using vaccine strategies to specific self-reactive T cells and B cells. As the list of diseases that are potential targets for treatment by immunological approaches increases, modulators for the immune competent cells are in greater demand. Improved methods for increasing or repressing immune responses, while following safe guidelines for use in humans, represent a major unmet demand in modern medicine.

B Cell- and APC-Targeting Immunotherapies

The cell surface molecule CD40 is a member of the tumor necrosis factor receptor superfamily and is broadly expressed by immune, hematopoietic, vascular, epithelial, and other cells, including a wide range of tumor cells. CD40 itself lacks intrinsic kinase or other signal transduction activity, but rather mediates its diverse effects via an intricate series of downstream adapter molecules that differentially alter gene expression depending on cell type and microenvironment. As a potential target for novel cancer therapy, CD40 may mediate tumor regression through both an indirect effect of immune activation and a direct cytotoxic effect on the CD40-expressing tumor.

CD40 is best known as a critical regulator of cellular and humoral immunity via its expression on B lymphocytes, dendritic cells, and monocytes [Grewal and Flavell, Annu Rev Immunol., 16:111 (1998); van Kooten and Banchereau, J Leukoc Biol., 67:2 (2000)].

CD40 is also expressed on the cell surface of many other non-immune cells, including endothelial cells, fibroblasts, hematopoietic progenitors, platelets and basal epithelial cells [Grewal and Flavell, Annu Rev Immunol., 16:111 (1998); van Kooten and Banchereau, J Leukoc Biol., 67:2 (2000); Young et al., Immunol Today, 9:502(1998); Quezada et al., Annu Rev Immunol., 22:307 (2004)]. The CD40 ligand (CD40L), also known as CD154, is the chief ligand described for CD40 and is expressed primarily by activated T lymphocytes and platelets [van Kooten and Banchereau, J Leukoc Biol., 67:2 (2000); Armitage et al., Nature, 357:80 (1992)]. Atherosclerosis, graft rejection, coagulation, infection control, and autoimmunity are all regulated by CD40-CD40L interactions [Grewal and Flavell, Annu Rev Immunol., 16:111 (1998); van Kooten and Banchereau, J Leukoc Biol., 67:2 (2000)]. Many tumor cells also express CD40, including nearly 100% of B-cell malignancies and up to 70% of solid tumors.

Physiologically, CD40-induced signal transduction represents a major component of a process known as T-cell “help.” Ligation of CD40 on dendritic cells, for example, induces cellular maturation and activation as manifested by increased surface expression of co-stimulatory and MHC molecules, production of proinflammatory cytokines such as interleukin 12, and enhanced T-cell activation [van Kooten and Banchereau, J Leukoc Biol., 67:2 (2000); Quezada et al., Annu Rev Immunol., 22:307 (2004)]. CD40 ligation of resting B cells also increases antigen-presenting function and, in addition, induces proliferation and immunoglobulin class switching [van Kooten and Banchereau, J Leukoc Biol., 67:2 (2000); Quezada et al., Annu Rev Immunol., 22:307 (2004)]. Patients with germ line mutations in either CD40 or CD40L are markedly immunosuppressed, susceptible to opportunistic infections, and have deficient T-cell-dependent immune reactions, including IgG production, germinal center formation, and memory B-cell induction [Allen et al., Science, 259:990 (1993); Ferrari et al., Proc Natl Acad Sci USA, 98:12614 (2001); Etzioni A, Ochs H D. Pediatr Res., 56:519 (2004)]. Similar immunophenotypes are observed in mice deficient in CD40 or CD40L [Castigli et al., Proc Natl Acad Sci USA, 91:12135 (1994); Kawabe et al., Immunity, 1:167 (1994); Renshaw et al., J Exp Med., 180:1889 (1994); Xu et al., Immunity, 1:423 (1994)]. Agonistic CD40 antibodies have been shown to mimic the signal of CD40L and substitute for the function of CD4⁺ T lymphocytes in murine models of T-cell-mediated immunity [Bennett et al., Nature, 393:478 (1998); Ridge et al., Nature, 393:474 (1998); Schoenberger et al., Nature, 393:480 (1998)]. A key mechanism of this effect is thought to be CD40/CD40L-mediated activation of host dendritic cells. Growing evidence shows that stimulating APC with soluble CD40L or an agonistic anti-CD40 antibody can, at least in part, replace the need for T helper cells and generate antigen presenting cells (APCs) that are capable of priming cytotoxic T lymphocytes (CTL). To develop pharmacotherapeutic reagents targeting the CD40/CD40L pathway, series of soluble CD40L fusion proteins were disclosed. In one invention, CD40L was joined to antigens to deliver CD40-costimulation signal and antigens together to B cells and APCs (WO/2003/063899). In another invention, the conjugate of CD40L and a Toll-like receptor ligand, Flagellin, was created to trigger a synergistic activation signaling between CD40 and TLR5 in B cells and APCs (WO/2007/103048). All these innovations aimed to use CD40L fusion proteins as vaccines by stimulating antigen-specific B cell and APCs in vivo.

T Cell Targeting Immunotherapies

Two types of major T lymphocytes have been described, CD8⁺ cytotoxic lymphocytes (CTLs) and CD4⁺ helper cells (Th cells). CD8⁺ T cells are effector cells that, via the T cell receptor (TCR), recognize foreign antigens presented by class I major histocompatibility complex (MEW) molecules on, for instance, virally or bacterially infected cells. T helper cells are involved in both humoral and cell-mediated forms of effector immune responses. With respect to the humoral or antibody immune response, antibodies are produced by B lymphocytes through interactions with Th cells. Specifically, extracellular antigens, such as circulating microbes, are taken up by specialized APCs, processed, and presented in association with class II MEW molecules to CD4⁺ Th cells. These Th cells in turn activate B lymphocytes, resulting in antibody production. In contrast, the cell-mediated immune response functions to neutralize microbes that inhabit intracellular locations after infection of a target cell.

According to the two-signal model, optimal activation of antigen-specific T lymphocytes requires specific antigen recognition by lymphocytes (signal 1′) and additional signals (called ‘signal 2’ or co-stimulatory signals). In the absence of signal 2, lymphocytes fail to respond effectively and are rendered anergic. Signal 1 is provided by the interaction of the peptide-antigen-MHC complex with the TCR. Signal 2 is delivered to T cells by co-stimulatory cell surface molecules expressed on APCs. The process of co-stimulation is of therapeutic interest because the manipulation of co-stimulatory signals might provide a means either to enhance or to terminate immune responses.

The B7-1/B7-2-CD28/CTLA-4 pathway is the best-characterized T-cell co-stimulatory pathway and is crucial in T-cell activation and tolerance [Karandikar et al., J. Neuroimmunol. 89:10 (1998); Oosterwegel et al., Curr. Opin. Immunol. 11:294 (1999); Salomon and Bluestone, Annu. Rev. Immunol. 19:225 (2001); Sansom, Immunology, 101:169 (2000); Chambers et al., Annu. Rev. Immunol., 19:565 (2001)]. The B7-1/B7-2-CD28/CTLA-4 pathway includes two B7 family members, B7-1 (CD80) [Freeman et al., J. Exp. Med. 174:625 (1991); Freedman et al., J. Immunol. 137:3260 (1987); Yokochi et al., J. Immunol. 128:823 (1982)] and B7-2 (CD86) [Freeman et al., Science 262:909 (1993); Freeman, et al., J. Exp. Med. 178:2185 (1993); Azuma, et al. Nature 366:76 (1993)], that have dual specificity for two CD28 family members, the stimulatory receptor CD28 antigen-receptor signaling [Aruffo and Seed, Proc. Natl Acad. Sci. USA 84:8573 (1987); Gross et al., J. Immunol., 144:3201 (1990)], by promoting T-cell survival and thereby enabling cytokines to initiate T-cell clonal expansion and differentiation [Thompsonet et al., Proc. Natl. Acad. Sci. USA 86:1333 (1989); Lucas et al., J. Immunol., 154:5757 (1995); Shahinian et al., Science 261:609 (1993); Sperling et al., J. Immunol., 157:3909 (1996); Boise et al., Immunity 3:87 (1995)]. CD28 also optimizes the responses of previously activated T cells, promoting interleukin 2 (IL-2) production and T-cell survival.

Several members of the tumor necrosis factor receptor (TNFR) family function as co-stimulatory receptors after initial T cell activation. These include CD27, 4-1BB (CD137), OX40 (CD134), HVEM, CD30 and GITR [reviewed in Watts, Annu Rev Immunol., 23:23 (2005)].

To develop immunotherapeutic reagents targeting T cells, soluble co-stimulatory receptor extracellular fragments, soluble ligand extracellular fragments, fusion proteins or agonistic antibodies against receptors or specific ligands have been studied. Alternatively, un-agonistic soluble ligands or un-agonistic antibodies have been used to block co-stimulatory receptor signaling. Either by increasing or reducing the extent of T cell costimulation, the use of ligand-fusion proteins or antibodies has shown pharmaceutical benefits to diseases including autoimmune diseases, proliferative disorders such as cancer, or infectious diseases.

Death Receptor “Fas” and Disease Therapy

Fas (CD95), a member of the tumour-necrosis factor receptor (TNFR) superfamily, was originally described as a lymphocyte receptor that can induce apoptosis [Yonehara et al., J. Exp. Med., 169:1747 (1989); Trauth et al., Science, 245:301 (1989)]. Fas is expressed in many types of tissue including glia cells, neurons and neuronal cell lines [Shinohara et al., Cancer Res., 60:1766 (2000); Gomez et al., J. Neurosci. Res., 63:421 (2001); Becher et al., Neurosciences, 84:627 (1998); Raoul et al., J. Cell Biol. 147:1049 (1999); Raoul et al., Neuron, 35:1067 (2002); Matsushita et al. J. Neurosci. 20:6879 (2000); Cheema et al., J. Neurosci., 19:1754 (1999)]. The interaction between CD95 (Fas) and its ligand (Fas-ligand, or FasL) functions to limit the duration of the immune response and/or life-span of activated lymphocytes. Apoptosis induced by Fas-FasL binding serves to clear activated self-reactive lymphocytes. Problems caused by altering this pathway have been demonstrated in animals with defects in Fas↔Fas-ligand interactions. Mice having mutations, which inactivate Fas or FasL, develop numerous disorders including autoimmune pathology resembling that seen in patients with rheumatoid arthritis or systemic lupus. It has been demonstrated that injection of FasL-expressing virus into the joints of mice with collagen-induced-arthritis, results in apoptosis of synovial cells and relief of arthritis symptoms [Zhang et al., in J. Clin. Invest., 100:1951 (1997)]. Expression of the Fas ligand reduces the number of activated inflammatory cells, which play a role in the pathogenesis of autoimmune disease. Therefore, a gene therapy strategy for introducing FasL into the joints of rheumatoid arthritis patients could function to improve disease pathology by leading to destruction of the infiltrating mononuclear cells.

Soluble Fas ligand and receptor have also been shown to be associated with tissue damage and other adverse effects. Administering an agonistic anti-Fas antibody resulted in organ damage to mice [Galle et al., J. Exp. Med. 182:1223 (1995)]. Mice injected intraperitoneally with the agonistic antibody died within several hours, and analyses revealed that severe liver damage by apoptosis was the most likely cause of death.

Fas engagement by FasL, or by antibodies against Fas, initiates binding of the intracellular death domain of Fas to an adaptor protein, the Fas-associated death domain (FADD), which couples Fas to the caspase cascade. Caspase 8 (also known as FADD-like interleukin-1 converting enzyme; FLICE) is the most upstream caspase in the apoptosis pathway, and its cleavage is a hallmark of Fas-induced death [Nagata, Cell 88: 355 (1997); Medema et al. EMBO J. 16:2794 (1997)]. Fas-mediated death signals can be inhibited by the FLICE inhibitory protein (FLIP), which blocks caspase 8 binding to FADD [Irmler et al. Nature 388:190 (1997)]. The activation of a cascade of successive caspase cleavages finally results in the activation of endonucleases that catalyse DNA breakdown into nucleosome-sized fragments, a characteristic feature of apoptosis [Nagata, Cell 88:355 (1997)].

In addition to apoptosis, Fas has been reported to mediate diverse proliferative and regenerative functions, including co-stimulatory signalling during T-cell activation [Alderson et al. J. Exp. Med., 178:2231 (1993); Alderson et al. Int. Immunol. 6:1799 (1994); Desbarats et al. Proc. Natl. Acad. Sci. USA 96:8104 (1999)], induction of angiogenesis [Biancone et al. J. Exp. Med. 186:147 (1997)], and liver regeneration after partial hepatectomy [Desbarats and Newell, Nature Med. 6:920 (2000)].

In contrast to the well-characterized apoptotic pathway, relatively little is known about the signalling pathways involved in Fas-mediated growth induction, although Fas has been shown to activate the extracellular-signal regulated kinase (ERK) pathway [Trauth et al., Science 245:301 (1989)]. ERK, a serine/threonine kinase activated by mitogen-activated protein kinase (MAPK)/ERK kinase (MEK1), mediates the cellular response to many different growth and differentiation factors [reviewed in Fukunaga and Miyamoto, Mol. Neurobiol., 16:79 (1998)]. Notably, activation of ERK prevents Fas-induced apoptosis and, conversely, inhibition of ERK prevents Fas-induced proliferation, suggesting that the MEK1/ERK pathway is involved in the transduction of Fas-mediated growth signals [Trauth et al., Science 245:301 (1989); Holmstrom et al., EMBO J., 19:5418 (2000); Kataoka, et al. Curr. Biol., 10:640 (2000)]. At present, it is not clear how Fas engagement in T-cell co-stimulation, and in the regeneration of liver and nerves, bypasses an apoptotic signal and promotes a regenerative or co-stimulation signal.

Soluble Fas-ligands has been useful reagents to induce pathological cell-specific cell death. For example, a fusion protein that connected interleukin-2-IgFc-FasL was used to kill auto-reactive T cells in autoimmune disease therapy [Bulfone-Paus et al., Transplantation. 69:1386 (2000)]. In a similar approach, the fusion protein of CD40 extracellular domain and FasL extracellular domain, CD40-FasL, showed that cell death is contingent on the binding of CD40 to CD40L expressed on target cells [Siegmund et al., J Mol Med. 84:785(2006)]. A fusion construct comprised of VEGF and FasL was found to effectively kill cancer cells by a synergistic effect between VEGF signaling and Fas signaling (WO/2007/022273). Additionally, a fusion protein containing a DC20-specific antibody fragment and soluble FasL, ScFvRit:sFasL [Bremer et al., Cancer Res., 68:597 (2008)], was applied to non-Hodgkin lymphoma and B cell chronic lymphocytic leukemia. This fusion protein efficiently activated CD20 signaling and Fas cell death signaling, resulting in a far superior proapoptotic activity, compared with co-treatment with anti-CD20 antibody (rituximab) and soluble FasL. Therefore Fas ligand-based fusion proteins have shown promising results in the field of autoimmune diseases and cancer therapy by stimulating Fas-induced death signaling in pathological cells. Fas-associated regenerative or co-stimulation signaling has not been exploited for target cell specific therapy.

Immune Suppressive Molecules

Traditionally, various steroids and inhibitors that block the cell activation and growth signaling, such as FK506 or Rapamycin, are broadly used. Some drawbacks to the use of these reagents are that they are not specific to lymphocytes and their use is often accompanied by serious side effects. The first therapeutic agents (immune suppressors) were mostly non-specific and inhibited cellular proliferation [Van Assche et al., Curr Opin Gastroenterol. May 5 (2009); Arias et al., Transplant Rev (Orlando) 23:94 (2009); Ng et al., Front Biosci., 14:1627 (2009)]. These treatments generally led to serious side effects due to intrinsic lack of pharmacospecificity. Later, cyclosporin A (CsA) was the first of a new generation of immunosuppressants with a ‘site-specific’ mode of action. Mechanistically, CsA mediates its in vivo effects by repressing lymphocyte activation at an early stage. Due to a low degree of myelotoxicity, CsA was considered as an attractive therapeutic drug in clinical transplantation for inhibiting lymphocytic activities without affecting either phagocytosis or migration of the reticulo-endothelial system. In 1978, CsA was tested clinically and due to its strong efficacy was used worldwide in a majority of the transplant centers to maintain graft survival post surgery [Goumenos, Expert Opin Pharmacother. 9:1695 (2008); Beauchesne, Drug Dev Ind Pharm. 33:211 (2007)]. In the meantime, much work has been put into the design of new therapeutic strategies that would present lower side effects but retain substantial efficacy. Based on research on the antigen specific T cell activation by TCR and in the recent application of the non-stimulatory CD3-specific humanized antibody (Alegre et al., J. Immunol., 155:1544 (1995), blockade of co-stimulatory receptors (i.e. CD28) with CTLA4-Ig and CD40 with anti-CD40L antibody has been attempted. Human-specific humanized non-activating anti-CD3 antibody (teplizumab) was FDA approved to prevent the T cell response that causes T cell immune deficiencies in human.

Thus far, inhibition of co-stimulatory receptors or T cell receptors (by non-activating anti-CD3 antibody) has been partially effective in inducing antigen-specific immunological tolerance.

APCs and T cells, or between T cells and B cells are tightly regulated by cell surface receptors and their counter-receptors (ligands). Therefore various techniques and reagents to facilitate or repress major receptor interactions and their signaling mechanisms have been developed for disease therapeutic purposes. Receptor agonistic or blocking antibodies, soluble extracellular domain of ligand fusion proteins, soluble death receptor ligand fusion proteins and mitogenic or immunosuppressive substances of bacterial or plant origin as described supra showed levels of efficacy beneficial to the disease therapy, although the toxic side effects are often accompanied by strong pharmacotherapeutic efficacy.

The present invention provides methods to develop powerful target cell-specific immune-stimulating fusion proteins, which could lead to the effective immunotherapy of various diseases.

SUMMARY OF THE INVENTION

This invention is based on the discovery that nucleic acid constructs that encode a minimum of (i) one functional moiety of the extracellular domain of the TNF/TNF receptor (TNFR) family agonist or the extracellular domain of the immunoglobulin family agonist or cytokine family, (ii) a second functional moiety of the extracellular domain of the TNF/TNFR family agonist or a functional moiety of the extracellular domain of the immunoglobulin family agonist or cytokine family, (iii) an immunoglobulin Fc domain (designated as IgGFc or IgFc in the text) (optional) and (iv) an antigen and the corresponding polypeptide, which the corresponding soluble fusion peptides expressed thereby (optional). A construct containing these components, when cultured with immune cells, will elicit a de novo effect to cause cell activation (i.e. cell proliferation, cytokine expression or immunoglobulin production in T cells and B cells).

This invention is also based on the discovery that the nucleic acid constructs or the expressed soluble fusion polypeptide, when administered to a host, elicits a de novo effect on immunity (i.e. increased or repressed B cell immunity and T cell immunity responses).

The function of the nucleic acids or soluble fusion protein is termed as a “synergistic” de novo effect on immunity. Specifically, the intended effect of the nucleic acid or soluble fusion protein construct is a significantly increased immune response relative to when either of the respective agonistic polypeptides contained therein are administered alone.

Particularly, this invention provides nucleic acid constructs containing genes encoding soluble fusion proteins which comprises (i) a CD40 ligand, a Fas ligand extracellular domain and an immunoglobulin IgG Fc domain [Yoshida et al., Vaccine 28: 3688 (2010)], (ii) a CD28 ligand (B7-2), a Fas ligand extracellular domain and an immunoglobulin IgG Fc domain, (iii) a OX40 ligand, a 4-1BB ligand extracellular domain and an immunoglobulin IgG Fc domain, (iv) a CD40 ligand, a ICOS extracellular domain and an immunoglobulin IgG Fc domain, (v) a NGFβ ligand, a Fas ligand extracellular domain and an immunoglobulin IgG Fc domain, (vi) an IL-2 ligand, a Fas ligand extracellular domain and an immunoglobulin IgG Fc domain. The fusion proteins will preferably elicit a de novo effect to cause immune cell activation relative to when any of the respective agonistic polypeptides contained therein are administered alone.

As described in detail infra, these nucleic acid constructs or the corresponding encoded fusion polypeptides may be administered to a host in need of such treatment as a means of:

generating enhanced (significantly greater) primary and memory B cell responses relative to immunization with antigen alone

generating enhanced (significantly greater) primary and memory CD4⁺ and/or CD8⁺ T cell responses relative to immunization with antigen alone

inducing repressed (significantly lower) antigen-specific B cell and/or T cell responses relative to immunization with antigen alone

These nucleic acid constructs or the corresponding encoded fusion polypeptides may be used in treating any disease or condition in which the above-identified enhanced or repressed humoral and cellular immune responses are required therapeutically. This applies specifically to infectious diseases, proliferative disorders such as cancer, allergies, inflammatory disorders, and other chronic diseases where enhanced cellular immunity is required. Alternatively, these reagents can be used in cases where repressed cellular immunity is required, such as organ transplant rejection, autoimmune disorders and graft versus host response.

As described in detail infra, DNA constructs may comprise linear DNA, a bacterial plasmid, a viral vector such as adenoviral, baculovirus, or other viral vectors commonly used for gene therapy. Additionally, as described infra these DNA constructs or fusion polypeptides may comprise extracellular domains of other TNF/TNFR family member agonists, immunoglobulin family member agonists and cytokines. Examples of TNF/TNFR family member agonists include Fas, Fas ligand, CD27, CD30 ligand, HVEM, TROY, RELT, TNF-alpha, TNF-beta, CD70, RANK ligand, LT-alpha, LT-beta, GITR ligand and LIGHT. Examples of immunoglobulin family member agonists include B7-1, B7-2, CD28, CTLA4, ICOS and ICOS-ligand. Further, these DNA constructs may comprise genes encoding antigens of bacterial/viral pathogens and tumor specific proteins. Examples of bacterial and viral antigens include human immunodeficiency virus-1 (HIV-1)env, HIV-lpol, HIV-lgag. Examples of tumor-specific antigens include human melanoma-associated glycoprotein p9′7, MART-1 and Her2/neu breast cancer antigen.

Various other features and advantages of the present invention should become readily apparent with reference to the following description, definitions, examples, claims and appended Figures. In several places throughout the specification guidance is provided through lists of examples. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic diagram of the presumed structure of the multi-ligand fusion protein and the interaction with target cells to induce cell activation.

FIG. 2A-C. Registered sequence of CD40 ligand of human origin (SEQ ID NO: 1).

FIG. 3A-C. Registered sequence of oncostatin M of human origin (SEQ ID NO: 3).

FIG. 4A, B. Registered sequence of IgG1 Fc of human origin (SEQ ID NO: 5).

FIG. 5A-C. Nucleotide sequence and amino acid sequence of the CD40L-IgFc fusion protein (SEQ ID NO: 7).

FIG. 6A-C. Registered sequence of Fas ligand of human origin (SEQ ID NO: 9).

FIG. 7A-C. Nucleotide sequence and amino acid sequence of the FasL-IgFc fusion protein (SEQ ID NO: 11).

FIG. 8A-D. Nucleotide sequence and amino acid sequence of the CD40L-FasL-IgFc fusion protein (SEQ ID NO: 13).

FIG. 9. Western blotting analysis of CD40L-FasL-IgFc. A: anti-human IgG, B: anti-human FasL.

FIG. 10. Study of Fas binding by CD40L-FasL-IgFc.

FIG. 11. Proliferation of human PBMC induced with CD40L-FasL-IgFc.

FIG. 12. Non-T cell-specific stimulation by CD40L-FasL-IgFc.

FIG. 13. Effects of Polymixin B pre-absorption on the stimulation by CD40L-FasL-IgFc.

FIG. 14. Reduction of CD40L-FasL-IgFc-induced activation by signaling inhibitors.

FIG. 15. Induction of IgG secretion in PBL cells stimulated with CD40L-FasL-IgFc.

FIG. 16. The expression of PRDI-BF1 in response to CD40L-FasL-IgFc stimulation.

FIG. 17. Adjuvant activity of CD40L-FasL-IgFc in mice against OVA.

FIG. 18. Adjuvant activity of CD40L-FasL-IgFc in mice against Influenza hemaglutinin.

FIG. 19. Adjuvant activity of Plasmid DNA of CD40L-FasL-IgFc in mice against Influenza hemaglutinin.

FIG. 20. Adjuvant activity of CD40L-FasL-IgFc in mice against Influenza hemaglutinin-specific T cell response. A: total T cell response; B: CD8⁺ T cell response.

FIG. 21A-C. Registered sequence of B7-2 of human origin (SEQ ID NO: 15).

FIG. 22A-D. Nucleotide sequence and amino acid sequence of the B7-2-FasL-IgFc fusion protein (SEQ ID NO: 17).

FIG. 23A-C. Nucleotide sequence and amino acid sequence of the B7-2-IgFc fusion protein (SEQ ID NO: 19).

FIG. 24. Western blotting analysis of B7-2-FasL-IgFc. A: anti-human IgG, B: anti-human FasL.

FIG. 25. Study of Fas binding by B7-2-FasL-IgFc.

FIG. 26. Activation of CD4⁺ and CD8⁺ T cells by B7-1-FasL-IgFc.

FIG. 27. B7-2-FasL-IgFc stimulates IL-2 production of T cells.

FIG. 28. Induction of T-bet by B7-2-FasL-IgFc activation.

FIG. 29. Reduction of B7-2-FasL-IgFc-induced activation by signaling inhibitors.

FIG. 30. Immunosuppressant activity of B7-2-FasL-IgFc in mice against Influenza hemaglutinin.

FIG. 31. Suppressant activity of plasmid DNA of B7-2-FasL-IgFc in mice against Influenza hemaglutinin.

FIG. 32. Immunosuppressant activity of B7-2-FasL-IgFc in mice against Influenza hemaglutinin-specific T cell response. A: total T cell response; B: CD8⁺ T cell response.

FIG. 33. The repression of SEB-induced splenomegaly by co-administrated Plasmid DNA of B7-2-FasL-IgFc.

FIG. 34A-C. Registered sequence of the OX40 ligand of human origin (SEQ ID NO: 21).

FIG. 35A-C. Registered sequence of the 4-1BB ligand of human origin (SEQ ID NO: 23).

FIG. 36A-D. Nucleotide sequence and amino acid sequence of the OX40L-4-1BBL-IgFc (SEQ ID NO: 25).

FIG. 37A-C. Nucleotide sequence and amino acid sequence of the OX40L-IgFc (SEQ ID NO: 27).

FIG. 38A-C. Nucleotide sequence and amino acid sequence of the 4-1BBL-IgFc (SEQ ID NO: 29).

FIG. 39. Western blotting analysis of OX40L-4-1BBL-IgFc. A: anti-human IgG, B: anti-human FasL.

FIG. 40. Activation of PBMCs by OX40L-4-1BBL-IgFc.

FIG. 41. Adjuvant activity of OX40L-4-1BBL-IgFc/PCIneo in mice against Influenza hemaglutinin.

FIG. 42. Adjuvant activity of OX40L-4-1BBL-IgFc in mice against Influenza hemaglutinin-specific T cell response. A: total T cell response; B: CD8⁺ T cell response.

FIG. 43A-C. Registered sequence of ICOS of human origin (SEQ ID NO: 31).

FIG. 44A-D. Nucleotide sequence and amino acid sequence of CD40L-ICOS-IgFc (SEQ ID NO: 33).

FIG. 45. Western blotting analysis of CD40L-ICOS-IgFc. A: anti-human IgG, B: anti-human FasL.

FIG. 46. Adjuvant activity of Plasmid DNA of CD40L-ICOS-IgFc in mice against Influenza hemaglutinin.

FIG. 47. Adjuvant activity of CD40L-ICOS-IgFc/PCIneo in mice against Influenza hemaglutinin-specific T cell response. A: total T cell response; B: CD8⁺ T cell response.

FIG. 48A, B. Registered sequences of NGFβ of human origin (SEQ ID NO: 35).

FIG. 49A-C. Nucleotide sequence and amino acid sequence of the NGFβ-FasL-IgFc fusion protein (SEQ ID NO: 37).

FIG. 50. Western blotting analysis of NGFβ-FasL-IgFc. A: anti-human IgG, B: anti-human FasL.

FIG. 51. Adjuvant activity of Plasmid DNA of NGFβ-FasL-IgFc in mice against Influenza hemaglutinin.

FIG. 52. Adjuvant activity of NGFβ-FasL-IgFc/PCIneo in mice against Influenza hemaglutinin-specific T cell response. A: total T cell response; B: CD8⁺ T cell response.

FIG. 53A, B. Registered sequence of IL-2 of human origin (SEQ ID NO: 39).

FIG. 54A-D. Nucleotide sequence and amino acid sequence of IL-2-FasL-IgFc (SEQ ID NO: 41).

FIG. 55. Western blotting analysis of IL-2-FasL-IgFc. A: anti-human IgG, B: anti-human FasL.

FIG. 56. Adjuvant activity of Plasmid DNA of IL-2-FasL-IgFc in mice against Influenza hemaglutinin.

FIG. 57. Suppressant activity of IL-2-FasL-IgFc/PCIneo in mice against Influenza hemaglutinin-specific T cell response.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that the soluble protein expression products of fusion cDNA constructs, composed of a co-stimulation ligand extracellular domain linked with a Fas death receptor ligand extracellular domain and immunoglobulin Fc domain, stimulates human lymphocytes to proliferate and produce cytokines rather than inducing cell death or cell co-stimulation.

One embodiment of the invention provides cDNA constructs encoding a novel synergistic agonistic polypeptide combination comprising (i) a cDNA encoding a specific TNF family agonistic extracellular domain polypeptide (i.e. CD40 ligand extracellular domain) and (ii) a cDNA encoding a different TNF family agonistic extracellular domain specific to Fas agonist (Fas ligand extracellular domain). Optional components of these constructs include (iii) cDNA encoding an immunoglobulin Fc domain and (iv) the cDNA encoding the desired antigen polypeptide. The soluble expression products of these gene constructs, designated as CD40 ligand (L)-Fas ligand (L)-IgFc (SEQ ID NO: 13), when cultured with a host cells stimulate CD40 expressing cells, inducing cell proliferation and cytokine production. These DNA constructs, either the vectors themselves or the purified protein they encode, when administered to a host may be used to generate enhanced humoral and cellular antigen-specific immune responses.

The present invention provides expression vectors and host cells containing a cDNA construct encoding this novel synergistic agonistic polypeptide combination comprising (i) a cDNA encoding a specific TNF family agonistic extracellular domain polypeptide (i.e. CD40 ligand extracellular domain) and (ii) a cDNA encoding a different TNF family agonistic extracellular domain specific to Fas (Fas ligand extracellular domain). Optional components of these constructs include (iii) cDNA encoding an immunoglobulin Fc domain and (iv) cDNA encoding the desired antigen against which enhanced antigen specific cellular immune response is a desirable outcome.

Another embodiment of the present invention provides DNA constructs encoding a novel synergistic agonistic polypeptide combination comprising (i) a cDNA encoding a specific immunoglobulin family agonistic extracellular domain polypeptide, preferably a CD28 agonist (i.e. B7-2 extracellular domain), and (ii) a cDNA encoding a different agonistic TNF family polypeptide extracellular domain of Fas agonist (Fas ligand extracellular domain). Optional components of these constructs include (iii) a cDNA encoding an immunoglobulin Fc domain and (iv) a cDNA encoding the desired antigen to which desired antigen-specific cellular immune response are elicited. The expression products of these gene constructs, designated as B7-2-FasL-IgFc (SEQ ID NO: 17), stimulate CD28 expressing cells resulting in the induction of cell proliferation and cytokine production. These DNA constructs, vectors containing or the expression product of these DNA constructs, when administered to a host, may be used to generate enhanced or repressed antigen-specific humoral or cellular immune responses.

The present invention further provides expression vectors and host cells containing a cDNA construct encoding novel synergistic agonistic polypeptide combination comprising (i) a cDNA encoding a specific immunoglobulin gene family agonistic extracellular domain polypeptide, preferably a CD28 agonist (B7-2 extracellular domain) and (ii) a cDNA encoding a different agonistic TNF family polypeptide extracellular domain of Fas agonist (Fas ligand extracellular domain). Optional components of these constructs include. Optional components of these constructs include (iii) cDNA encoding an immunoglobulin Fc domain and (iv) cDNA encoding the desired antigen against which repressed antigen specific cellular immune response are desirable outcome.

Another embodiment of the present invention provides cDNA constructs encoding a novel synergistic agonistic polypeptide combination comprising (i) a cDNA encoding a specific TNF family agonistic extracellular domain polypeptide, preferably a OX40 agonist (OX40 ligand extracellular domain) and (ii) a cDNA encoding a different TNF family agonistic extracellular domain of 4-1BB agonist (4-1BB ligand extracellular domain). Optional components of these constructs include (iii) cDNA encoding an immunoglobulin Fc domain and (iv) cDNA encoding the desired antigen. The expression products of these gene constructs, designated as OX40L-4-1BBL-IgFc (SEQ ID NO: 25), when cultured with a host cell, stimulate OX40 and 4-1BB expressing cells resulting in activation of T cells and regulatory T cells inducing cell proliferation and cytokine productions. These cDNA constructs, vectors containing or the protein expression product of these constructs, when administered to a host, may be used to generate enhanced antigen-specific humoral and cellular immune responses.

The present invention further provides expression vectors and host cells containing a cDNA construct encoding novel synergistic agonistic polypeptide combination comprising (i) a cDNA encoding a specific TNF family agonistic extracellular domain polypeptide, preferably a OX40 agonist (OX40 ligand extracellular domain) and (ii) a cDNA encoding a different TNF family agonistic extracellular domain of 4-1BB agonist (4-1BB ligand extracellular domain). Optional components of these constructs include (iii) cDNA encoding an immunoglobulin Fc domain and (iv) desired antigen to which enhanced antigen-specific cellular immune response is a desirable outcome.

Another embodiment of the present invention provides DNA constructs encoding a novel synergistic agonistic polypeptide combination comprising (i) a cDNA encoding a specific TNF family agonistic extracellular domain of specific CD40 agonist (CD40 ligand extracellular domain), and (ii) a cDNA encoding a specific immunoglobulin family receptor extracellular domain polypeptide, preferably a ICOS (i.e. ICOS extracellular domain). Optional components of these constructs include (iii) a cDNA encoding an immunoglobulin Fc domain and (iv) a cDNA encoding the desired antigen to which desired antigen-specific cellular immune response are elicited. The expression products of these gene constructs, designated as CD40L-ICOS-IgFc (SEQ ID NO: 33), stimulate ICOS ligand expressing cells resulting in the induction of cell proliferation and cytokine production. These DNA constructs, vectors containing or the expression product of these DNA constructs, when administered to a host, may be used to generate enhanced or repressed antigen-specific humoral or cellular immune responses.

The present invention further provides expression vectors and host cells containing a cDNA construct encoding novel synergistic agonistic polypeptide combination comprising (i) a cDNA encoding a specific different TNF family agonistic extracellular domain polypeptide of specific CD40 agonist (CD40 ligand extracellular domain), and (ii) a cDNA encoding a specific immunoglobulin family receptor extracellular domain polypeptide, preferably a ICOS (i.e. ICOS extracellular domain). Optional components of these constructs include (iii) a cDNA encoding a immunoglobulin Fc domain and (iv) a cDNA encoding the desired antigen against which repressed antigen specific cellular immune response are desirable outcome.

Another embodiment of the present invention provides cDNA constructs encoding a novel synergistic agonistic polypeptide combination comprising (i) a cDNA encoding a specific, neuro growth factor (NGF) family polypeptide, preferably NGFβ and (ii) a cDNA encoding a specific TNF family agonistic extracellular domain polypeptide of Fas agonist (Fas ligand extracellular domain). Optional components of these constructs include (iii) cDNA encoding an immunoglobulin Fc domain and (iv) cDNA encoding the desired antigen to which enhanced antigen-specific cellular immune responses are a desired outcome. The expression products of these gene constructs, designated as NGF-FasL-IgFc (SEQ ID NO: 37), when cultured with a host cells in vitro preferably, stimulate NGF receptor expressing cells (i.e. neuronal cells and B cells) resulting in the induction of cell proliferation, cell differentiation and cytokine production. These cDNA constructs, vectors containing or the expression product of these DNA constructs, when administered to a host may be used to generate (i) growth promotion of nerve cells and (ii) enhanced antigen-specific humoral and cellular immune responses.

The present invention further provides expression vectors and host cells containing a cDNA construct encoding novel synergistic agonistic polypeptide combination comprising (i) a cDNA encoding a specific NGF family polypeptide, preferably NGFβ and (ii) a cDNA encoding a specific TNF family agonistic extracellular domain polypeptide of Fas agonist (Fas ligand extracellular domain). Optional components of the constructs include (iii) cDNA encoding an immunoglobulin Fc domain and (iv) cDNA encoding the desired antigen to which enhanced antigen specific cellular immune response are targeted.

Another embodiment of the present invention provides cDNA constructs encoding a novel synergistic agonistic polypeptide combination comprising (i) a cDNA encoding a specific interleukin 2 family agonistic polypeptide, preferably a interleukin 2 and (ii) a cDNA encoding a specific TNF family agonistic extracellular domain polypeptide of Fas agonist (Fas ligand extracellular domain). Optional components of these constructs include (iii) cDNA encoding an immunoglobulin Fc domain and (iv) desired antigen to which enhanced antigen-specific cellular immune response are desirable outcome. The protein expression products of these gene constructs, designated as IL-2-FasL-IgFc (SEQ ID NO: 41) when cultured with a host cells in vitro stimulate IL-2 receptor expressing cells resulting in activated T cells and B cells and the induction of cell proliferation and cytokine production. These cDNA constructs, vectors containing or the expression product of these cDNA constructs, when administered to a host, may be used to generate enhanced antigen-specific humoral and repressed cellular immune responses.

The present invention further provides expression vectors and host cells containing a cDNA construct encoding novel synergistic agonistic polypeptide combination comprising (i) a cDNA encoding a specific interleukin 2 family agonistic polypeptide (i.e. interleukin 2 itself) and (ii) a cDNA encoding a specific TNF family agonistic extracellular domain polypeptide of Fas agonist (Fas ligand extracellular domain). Optional components of these constructs also include (iii) cDNA encoding an immunoglobulin Fc domain and (iv) cDNA encoding the desired antigen to which enhanced antigen-specific cellular immune responses are a desirable outcome.

In addition, the invention provides methods of using vectors and host cells to produce a composition containing the novel synergistic conjugates (i) CD40 ligand/Fas ligand [Yoshida et al., Vaccine 28: 3688 (2010)], (ii) B7-2/Fas ligand, (iii) OX40 ligand/4-1BB ligand, (iv) CD40 ligand/ICOS, (v) NGFβ/Fas ligand or (vi) IL-2/Fas ligand. In each of the 6 vectors described, there is an option to link to an Fc/antigen polypeptide conjugate.

Further, the invention provides methods of administering the DNA or fusion proteins into a host in which an antigen-specific immune response is required to be elicited or repressed. For example, administration of these reagents to a subject with a chronic disease such as cancer, an infectious or autoimmune disease, or an allergic disorder, may be beneficial.

The invention also provides compositions comprising novel synergistic fusion constructs containing antigen polypeptide conjugates, which are suitable for administration to a host in order to elicit an enhanced antigen-specific humoral or cellular immune response.

The invention provides novel methods of immunotherapy comprising the administration of said novel synergistic fusion constructs of agonists-antigen polypeptide conjugate or a cDNA encoding said fusion protein polypeptide to a host in need of such treatment in order to elicit an enhanced antigen-specific humoral and cellular immune response. In preferred embodiments, these compositions and conjugates will be administered to a subject with or at risk of developing a cancer, an infection, particularly a chronic infectious diseases (i.e. involving a virus, bacteria or parasite) or an autoimmune, inflammatory or allergic condition. As an example, in the preferred embodiment described infra, the invention is used to elicit antigen-specific cellular immune responses against chronic viral infections such as type A Influenza virus and human immunodeficiency virus HIV-1. HIV-1 infection is a well-recognized example of a disease where protective immunity almost certainly will require the generation of potent and long-lived cellular immune responses against the virus.

Further, the invention provides novel methods of immunotherapy comprising the administration of said novel synergistic fusion proteins encoding agonists-antigen polypeptide conjugates or cDNA constructs encoding said fusion protein polypeptide to a host in need of such treatment in order to elicit a repressed antigen specific humoral and cellular immune response. In preferred embodiments, these compositions and conjugates will be administered to a subject with or at risk of developing an organ rejection reaction against transplanted allo or xeno organs (i.e. kidney, heart, liver) or autoimmune, inflammatory or allergic conditions.

As used herein the following terms shall have the meanings set forth. Otherwise all terms shall have the meaning they would normally be accorded by a person skilled in the relevant art.

The term “agonist” refers to a compound that in combination with a receptor can produce a cellular response. An agonist may be a ligand that directly binds to the receptor. Alternatively an agonist may combine with a receptor indirectly by for example (a) forming a complex with another molecule that directly binds to the receptor, or (b) otherwise resulting in the modification of another compound so that the other compound directly binds to the receptor. An agonist herein will typically refer to a TNF family agonist, TNFR family agonist, an immunoglobulin family agonist or a cytokine family agonist.

The term “antigen” herein refers to any substance that is capable of being the target of an immune response. An antigen may be the target, for example, of a cell-mediated and/or humoral immune response (i.e. immune cell maturation, production of cytokines, production of antibodies, etc.) when contacted with immune cells. Examples of antigens include bacterial, viral, fungal polypeptides, autoantigens, allergens, and the like.

The term “synergy” and variations thereof refers to activity such as immunostimulatory activity achieved when administering a combination of active agents that is greater than the additive activity of the active agents administered individually.

The term “conjugate” herein refers to a single molecule, typically a DNA fusion or polypeptide fusion that contains a plurality of agonists or genes encoding and optionally an antigen or gene encoding wherein each are directly or indirectly attached to one another, e.g., by the use of linkers, and wherein these agonists and antigen, if present, may be in any order relative to one another in the conjugate.

Herein “(i) CD40 ligand (CD40L), (ii) Fas ligand (FasL), (iii) B7-2, (iv) IL-2, (v) NGFβ, (vi) OX40 ligand (OX40L), (vii) 4-1BB ligand (4-1BBL) and (viii) ICOS” includes any polypeptide or protein that specifically recognizes and activates the (i) CD40 receptor, (ii) Fas receptor, (iii) CD28 receptor, (iv) IL-2 receptor, (v) NGF receptor, (vi) OX40 receptor, (vii) 4-1BB receptor and (viii) ICOS ligand respectively, and activates its biological activity. Preferably they are human corresponding proteins or derivatives or polymers or fragments thereof. Particularly the invention embraces CD40L, FasL, B7-2, IL-2, NGF, OX40L, 4-1BBL and ICOS proteins and fragments possessing at least 75-80% identity, more preferably at least 90%-95% sequence identity or more to the native polypeptide or a fragment thereof which recognize and activate the specific receptors. The amino acid sequence and corresponding nucleic acid sequence of each polypeptide is disclosed; CD40L in U.S. Pat. Nos. 6,410,711 and 5,962,406; FasL in U.S. Pat. No. 6,046,310, WIPO Patent No. WO/1997/033617, WO/1995/013293 and; B7-2 in U.S. Pat. No. 5,942,607; OX40L in U.S. Pat. No. 5,457,035; 4-1BBL in U.S. Pat. No. 6,838,262; IL-2 in Genbank No. NM_000586; NGFβ in GenBank No. NM_002506 and ICOS in GenBank No. NM_012092 all incorporated by reference in their entirety herein.

“TNF/TNFR” refers to a member of the tumor necrosis factor superfamily or the tumor necrosis factor receptor superfamily. Examples thereof include CD40, CD40L, 4-1 BB, 4-IBBL, CD27, CD70, CD30, CD30 ligand (CD153), OX40, OX-40L, Fas, FasL, TNF-alpha, TNF-beta, TNFR2, RANK, LT-beta, LT-alpha, HVEM. GITR, TROY, RELT, of any species and allelic variants and derivatives thereof.

The term “cytokine” is understood to mean any protein or peptide, analog or functional fragment thereof, which is capable of stimulating or inducing a pro survival effect against a pre-selected cell-type, for example, a lymphocyte or a monocyte, in a mammal. Accordingly, it is contemplated that a variety of cytokines can be incorporated into the cytokines of the invention. Useful cytokines include, for example, tumor necrosis factors (TNFs), interleukins (ILs), lymphokines (Ls), colony stimulating factors (CSFs), interferons (IFNs) including species variants, truncated analogs thereof which are capable of stimulating or inducing such pro survival responses. Useful neuro growth factors include, for example, NGFβ. Useful lymphokines include, for example, LT. Useful colony stimulating factors include, for example, GM-CSF and M-CSF. Useful interleukins include, for example, IL-2, IL-4, IL-5, IL-7, IL-12, IL-15 and IL-18. Useful interferons, include, for example, IFN-α, IFN-β and IFN-γ.

The terms “treatment” and “therapeutic method” refer to both therapeutic treatment and prophylactic/preventative measures. Those in need of treatment may include individuals already having a particular medical disorder as well as those who may ultimately acquire the disorder (i.e., those needing preventative measures).

The term “isolated” refers to a molecule that is substantially free of its natural environment. For instance, an isolated protein is substantially free of cellular material or other proteins from the cell or tissue source from which it is derived. The term “isolated” also refers to preparations where the isolated protein is sufficiently pure to be administered as a pharmaceutical composition, or at least 70-80% (w/w) pure, more preferably, at least 80-90% (w/w) pure, even more preferably, 90-95% pure; and, most preferably, at least 95%, 96%, 97%, 98%, 99%, or 100% (w/w) pure.

The term “co-stimulate” with reference to activated immune cells includes the ability of a co-stimulatory molecule to provide a second, non-activating receptor mediated signal (a “costimulatory signal”) that induces proliferation or effector function. For example, a co-stimulatory signal can result in cytokine secretion (i.e. in a T cell that has received a T cell-receptor-mediated signal).

As used herein the term “co-stimulatory molecule” includes molecules, which are present on (i) antigen presenting cells (i.e. B7-1, B7-2, B7RP-1, ICOSL, OX40L, 4-1BBL and/or related molecules that bind to co-stimulatory receptors (i.e., CD28, CTLA4, ICOS, OX40, 4-1BB and/or related molecules) on T cells, and (ii) T cells (i.e. CD40L, ICOS and/or related molecules that bind to costimulatory receptors (i.e. CD40, ICOSL and/or related molecules) on antigen presenting cells and B cells.

The term “soluble” includes molecules, such as co-stimulatory molecules, which are not cell associated. Soluble co-stimulatory molecules retain the function of the cell-associated molecules from which they are derived in that they are capable of binding to their cognate ligands on T cells and mediating signal transduction via (i) CD28, CTLA4, OX40, 4-1BB and/or related molecules on a T cell, and (ii) CD40, ICOSL and/or related molecules on antigen presenting cells and B cells, however, they are in soluble form (i.e. they are not membrane bound).

The term “fusion” or “fusion protein” as used herein refers to the combination of amino acid sequences in one polypeptide chain, preferably by in frame fusion of corresponding coding nucleotide sequences. In nature, the X and Y entities normally exist in separate proteins, which are brought together in the fusion protein of the invention. In the fusion protein of the present invention, the coding sequence of the first polypeptide (X) is fused in frame with the coding sequence of the second polypeptide (Y) either directly or through a linker. By “fused in frame”, it is meant that the expression of the fused coding sequences results in the fusion protein comprising both the first and the second polypeptides. This means, for example, that there is no translational terminator between the reading frames of the X and Y polypeptides. Even through the fusion between the X and Y entities can take place internally at any site, the Y entity is preferably fused to either the COOH or the NH2 terminus of the X entity (resulting in a fusion of the formula X—Y and Y-X respectively). As used herein, the term “directly” refers to a fusion of the polypeptides X and Y without a peptide linker in between (i.e. the codons encoding the X entity are contiguous with the codons encoding the Y entity). In addition, the fusion protein may also include further elements (Z) apart from X, Y and a linker, such as an initiator methionine, a signal peptide, an anchor polypeptide (e.g., immunoglobulin Fc domain) and an antigen polypeptide. Fusion proteins essentially consisting of X and Y, Z (optional) and a linker (optional) are preferred embodiments in the context of the present invention.

The fusion proteins encompassed by the present invention are not limited by the particular identity of X and Y, nor by the number of X and/or Y entities employed in the fusion protein. The X and the Y polypeptides are different (i.e. heterologous with respect to one another). The difference may be in terms of structure (i.e. below 40% identity between their respective amino acid sequences) and/or in terms of their respective biological activity (i.e. X and Y are involved in different pathways of the immune system). The X and Y entities involved in the fusion protein of the invention may individually originate (be obtained, isolated) from human or animal origin (i.e. canine, avian, bovine, murine, ovine, porcine, feline, simian and the like). The fusion protein may also comprise X and Y entities of diverse origins (i.e. X of human origin and Y of animal origin).

The current invention is based on the discovery in that the immunoglobulin IgG Fc domain-conjugated fusion protein comprising an extracellular domain of B7-2 (the ligand of CD28 T cell co-stimulation receptor, Fas antigen), and an extracellular domain of Fas-ligand (the ligand of cell death receptor), did not induce cell death in human Fas-apoptosis-sensitive Jurkat T cell line or human peripheral blood T lymphocytes. The net effect of this fusion protein, designated as B7-2-FasL-IgFc (SEQ ID NO: 17), was a strong activation of proliferation in peripheral blood T lymphocytes. The observed activation was comparable to that induced by the well-known T cell mitogen PHA. The binding of B7-2 with the CD28 co-stimulation receptor alone does not cause T cell proliferation or cytokine production, while treatment with Fas-ligand alone renders T lymphocytes highly susceptible to cell death. According to understanding, the strong cell activation by simultaneous stimulation of a co-stimulation receptor and another death receptor is not a predictable outcome. Indeed, the additive stimulation of Fas and CD28 by Fas- and CD28-specific specific monoclonal antibodies does not cause T cell activation. Similarly, the T cell activation was caused only by the B7-2-FasL-IgFc fusion protein and not by treatment with a mixture of the B7-2-IgFc (SEQ ID NO: 19) and FasL-IgFc (SEQ ID NO: 11) fusion proteins. A possible explanation for the strong activation in CD28⁺/Fas⁺ T cells is that B7-2 and FasL must be located in the close proximity to each other, as is the case when linked together in the B7-2-FasL-IgFc fusion protein. Interestingly, although B7-2-FasL-IgFc polypeptide induced a mitogenic response in T cells cultured in vitro, the administration of the polypeptide or the plasmid DNA in vivo resulted in the severe repression of humoral and cellular immune responses.

Since Fas is expressed in almost the entire population of lymphocytes, the specificity of B7-2-FasL-IgFc to T cells is presumed to be determined by the binding of the B7-2 domain CD28 on T cells. To investigate this further, the inventor asked if the replacement of B7-2 extracellular domain with an extracellular domain of the B cell costimulatory ligand (a CD40 ligand), could change the specificity of the resulting CD40L-FasL-IgFc fusion protein. Indeed, the treatment with this fusion protein, designated CD40L-FasL-IgFc, did not activate T lymphocytes but resulted in a strong mitogenic activity against B cells.

The experiments described supra demonstrated that a fusion protein, which conjugates “X” (an extracellular domain of a co-stimulatory receptor ligand) with “Y” (a Fas ligand extracellular domain) and “Z” (IgFc domain) possesses de novo mitogenic activity. This formula to create the mitogen fusion protein was further exploited by testing if both X and Y portions could be replaced with extracellular domains of other costimulation receptors (FIG. 1). When the ligand of T cell OX40 costimulatory receptor, and the ligand of 4-1BB T cell costimulatory receptor were used as the “X” and “Y” respectively, the resulting fusion protein (OX40L-4-1BBL-IgFc, SEQ ID NO: 25) acquired a strong mitogen activity in human peripheral blood mono nuclear cells. When stimulated by either OX40L-IgFc (SEQ ID NO: 27) or 4-BBL-IgFc (SEQ ID NO: 29) alone, however, no significant mitogen activity was observed.

In further studies, the fusion protein which conjugates “X” (the extracellular domain of CD40L with “Y” (the extracellular domain of ICOS, a costimulatory receptor of activated T cells) was constructed and designated CD40L-ICOS-IgFc (SEQ ID NO: 33). By replacing the Fas ligand with immunoglobulin family polypeptide, ICOS, the resulting fusion protein was expected to target B cells, which express both CD40 and ICOS ligand. When injected into mice along with influenza hemaglutinin (HA) antigens, the CD40L-ICOS-IgFc gene construct (SEQ ID NO: 33) caused a significantly increased HA-specific antibody response. The reason for this was that the CD40L-ICOS-IgFc plasmid DNA functioned as a DNA adjuvant. These results demonstrated that it is possible to create various functional fusion proteins with different target cell specificity by changing the combination of the “X” and “Y” components.

In another expansion, the fusion protein comprising NGFβ as “X” and FasL as “Y” was generated. This fusion protein, designated NGFβ-FasL-IgFc (SEQ ID NO: 37), was expected to target nerve cells and B cells as in previous reports showed that NGF0 could promote B cell growth. As expected, the fusion protein caused a strong B cell antibody response in vivo when the expression plasmid was co-administrated with influenza HA antigens. This fusion protein gene construct also induced a strong HA-specific CD8⁺ T cell response in mice co-administrated with the HA antigens.

Another fusion protein, in which interleukin 2 (IL-2) was inserted to the domain “X”, was designated IL-2-FasL-IgFc (SEQ ID NO: 41). Based on the properties of its conjugated proteins, IL-2-FasL-IgFc was expected to stimulate IL-2 receptor-positive T cells and B cell. Our data indicates that IL-2-FasL-IgFc treatment resulted in a repressive effect on cellular responses against the co-administrated influenza HA antigens in mice. Interestingly, the in vivo function of IL-2-FasL-IgFc was similar to that shown by B7-FasL-IgFc administration in that is also targeted T cells.

It has been long established that many receptors and their contra-receptors (ligands) expressed trigger bi-directional signaling. Thus receptor extracellular domains become ligands to the contra-receptors during receptor/contra-receptor-mediated cell-cell communication. Accordingly, many receptor extracellular domains would be available for “X” or “Y” in the fusion protein constructs described herein to create target cell-specific mitogens.

The provided examples of fusion proteins and their functions demonstrate the robust potential of the fusion proteins generated based on the construct of “X”-“Y”-IgFc paradigm. Moreover, IgG Fc could be replaced with other domains of immunoglobulins such as IgM Fc domain, which would result in the formation of pentamers of the fusion protein.

The beneficial characteristics of the fusion proteins in the current invention would include:

-   -   i) The fusion protein comprises the polypeptides derived from         extracellular domains of co-stimulation receptors, death         receptors and their ligands to activate T cells, B cells or         antigen presenting cells. This will allow lymphocytes still         capable of recognizing antigens by T cell antigen receptors or B         cell immunoglobulin receptors during the activation by fusion         protein mitogens.     -   ii) Following the recipe “X”-“Y”-IgFc, the target specific         mitogens could be customized for any target molecule by choosing         target cell-specific co-stimulatory or death receptor ligands.     -   iii) Following the recipe “X”-“Y”-IgFc, the desirable function         could be designed as a single molecule stimulation fusion         protein by choosing specific receptor ligands.

As described by example in the study of B7-2-FasL-IgFc and IL-2-FasL-IgFc, the administration of the fusion protein or expression plasmid DNA represses the in vivo immune response against co-administrated antigens. This feature of the fusion protein would be applicable to the disease therapies, in which there is a need to apply immunosuppressive treatment.

The fusion protein, B7-2-FasL-IgFc, could be used as is to cause adjuvant activity or repressant activity to the immune response in vivo against the co-administrated antigens.

The fusion protein, B7-2-FasL-IgFc, could be conjugated with antigen polypeptides by recombinant DNA techniques to create single molecule adjuvant-antigen vaccines or single molecule immunorepressant-antigen tolerance inducers.

The plasmid DNA consisting of the fusion protein, B7-2-FasL-IgFc, could be used as DNA adjuvant or DNA suppressant by in vivo administration.

These characteristics make the described the fusion protein, B7-2-FasL-IgFc, excellent immunological adjuvant or suppressant candidates, which would satisfy unmet demands in broad areas of medicine.

Some of the innovations aimed to apply the soluble death receptor ligand extracellular domain fusion proteins to target the death receptor of pathological cells, such as cancer cells or auto-reactive immune cells. In other instances, co-stimulatory receptor ligands were used to block the co-stimulatory receptor signaling. In addition, co-stimulatory receptor-specific monoclonal antibodies were often used to enhance the activation of T cells, B cells or antigen presenting cells as to cause adjuvant effects. In some cases, Fas ligand was conjugated with other functional molecules such as interleukin-2 or VEGF, in order to navigate the fusion protein to specific target cells and increase their sensitivity to cell death. In these examples, Fas ligand domains in the fusion constructs are carefully positioned to the C-terminal ends of the polypeptides to cause cell death in target cells following ligation with the endogenous Fas receptor. The current invention should be construed as unique because the manner in which Fas ligand domain is integrated does not result in cell death in Fas^(t) cells.

The in vivo repressor function by some fusion proteins and their plasmid DNAs could be valuable for the immune-suppressive therapy against both organ transplant rejection and autoimmune diseases. For example, B7-2-FasL-IgFc is an outstanding candidate for the induction of antigen-specific immunological tolerance in vivo—a function which no known current medicine can fulfill. Other than plant lectins (i.e. Con A and PHA) or bacterial enterotoxins (i.e. SEB and SEA), only few molecules are known to be T cell mitogens. Specific potential example is superagonistic anti-CD28 antibody [Beyersdorf et al., Ann Rheum Dis. 64:91 (2005)]. Recent studies of superagonistic anti-CD28 antibody (WO/2006/050949) showed the potential of desirable effects in vivo by increasing the activity of T regulatory cells, which repressed disease symptoms in animal autoimmune disease models [Beyersdorf et al., Ann Rheum Dis. 64:91 (2005)]. The administration of superagonistic anti-CD28 antibody to humans, however, resulted in serious toxic side effects in clinical trials [Suntharalingam et al., N Engl J Med., 355:1018 (2006)]. Thus antigen-specific T lymphocyte repressants remain in great demand. The B7-2-FasL-IgFc of the current invention could ease the shortage of the T lymphocyte mitogens usable to induce antigen-specific tolerance in vivo.

Immune adjuvant effects in vivo by fusion proteins and their plasmid DNAs of the current invention will be highly useful in the development of effective vaccines. Until now, the development of vaccine adjuvants has heavily weighed on the ligands for the Toll like receptor (TLR) family receptors. The stimulation of the TLR family receptors expressed by antigen presenting cells causes increased expression of ligands and cytokines in turn causing increased the T cell and B cell co-stimulation signaling. This co-stimulation signaling is crucial for the efficient vaccination by inducing T cell differentiation, T cell memory development, B cell differentiation and B cell antibody-affinity maturation. Therefore the TLR ligands indirectly facilitate the events essential for the efficient vaccination. The fusion proteins and their plasmid DNAs of the current invention will become very effective adjuvants by directly stimulating co-stimulation signaling in B cells and antigen presenting cells, thereby enhancing the CD8⁺ T cell memory and B cell immunoglobulin affinity maturation to a great extent.

Exemplification of the Invention with Model Fusion Proteins

Example 1

Methods and Materials

1. General Recombinant DNA Methods

Unless otherwise indicated, for generating nucleic acids encoding the fusion proteins of the present invention and for expression of the fusion proteins, routine techniques in the field of recombinant genetics were employed. Basic texts disclosing the general methods of use in this invention include Sambrook & Russell, Molecular Cloning, A Laboratory Manual (3rd Ed, 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al, eds., 1994-2009).

For proteins, sizes are given in kilodaltons (kDa) or number of amino acid residues. Proteins sizes are estimated from gel electrophoresis, protein sequencing, amino acid sequences deduced from cDNA, or from published protein sequences.

Oligonucleotides that are not commercially available can be chemically synthesized according to the solid phase phosphoramidite triester [Beaucage and Caruthers, Tetrahedron Letts. 22:1859-1862 (1981)], using an automated synthesizer [Van Devanter et al, Nucleic Acids Res. 12:6159 (1984)]. Purification of oligonucleotides is performed by either native acrylamide gel electrophoresis or by anion-exchange HPLC [Pearson and Reanier, J. Chrom. 255:137 (1983)]. The sequence of the cloned genes and synthetic oligonucleotides can be verified after cloning using the chain termination method for sequencing double-stranded templates [Wallace et al, Gene 16:21 (1981)].

2. Western Blotting

Antibodies against the extracellular domain of Fas ligand (sc-957), OX40 ligand (sc-11404), CD40 ligand (sc-978) and horse raddish peroxidase (HRP)-conjugated human IgG (sc-2923) were purchased from Santa Cruz Biotechnology. The ligand-specific antibodies were used for Western blotting at a final concentration of 1.0 μg/ml. The secondary antibodies used were goat anti-rabbit IgG horse HRP-conjugated antibody (sc-2054, Santa Cruz Biotechnology). Both secondary antibodies were used at a final concentration of 0.4 μg/ml. Bands were visualized using chemiluminescence (ECL reagents from Amersham).

3. Proliferation and ELISA Assays

Human lymphocytes were stimulated for 3 days in 96 U-bottom well plates with various concentrations of recombinant fusion proteins. In some experiments, a NF-κB inhibitor (SN50, 18 μM, Calbiochem, San Diego, Calif., USA) and a MEK1/2 inhibitor (PD98059, 1 μg/ml, Calbiochem) were added to the cell culture media. Cell proliferation was assessed by thymidine uptake assays. Cultured cells were pulsed with 1 μCi/well of [³H]thymidine (ICN, Costa Mesa, Calif., USA) for 18 hours and then harvested onto glass fiber filters. [³H]thymidine incorporation was measured by standard liquid scintillation counting techniques. In some experiments, HA antigen-specific cell proliferation response was assayed by the MTT assay [van de Loosdrecht et al., J. Immunol. Methods, 174:311(1994)], which is a direct measure of cell viability.

The relative concentrations of TNP-specific human IgG and IgM in supernatants from 4 day cultures were determined by ELISA assays. TNP₂₂-BSA (1 μg/ml) was used to coat assay plates, and alkaline phosphatase-conjugated anti-human IgG (Fab-specific) (Sigma) or anti-human IgM (μ-chain-specific) (Sigma) were used as the detection reagents. The concentration of IL-2 in fusion protein-stimulated culture supernatants was measured by human-IL-2-specific ELISA kit (Roche, Laval, QC, Canada). The lower limit of detection was 30 pg/ml in IL-2 assay.

4. Assay for PRDI-BF1, T-Bet and GATA3 RNAs Concentration

Using specific primers, PRDI-BF1 RNA was amplified by RT-PCR (20 cycles) from cytoplasmic RNA derived from PBL cells stimulated for three days with fusion proteins (0.1 μM). T-bet and GATA3 RNAs were amplified by RT-PCR (25 cycles) from cytoplasmic RNA derived from CD4⁺ T cells stimulated for five days with fusion proteins (0.1 μM). Each sample was subjected to electrophoresis on agarose gels and stained with ethidium bromide (EtBr) to detect amplified fragments: 594 bp for PRDI-BF1, 206 bp for T-bet and 131 bp for GATA3.

5. Vaccination of Mice

Balb/c mice (4-6 wks of age) were primed intra peritoneal (i.p.) route with 0.1 ml of protein antigens, OVA albumin (Sigma) or GSK VAXIGRIP (GlaxoSmithKline, Mississauga, Ontario, Canada), in the presence and absence of fusion protein or cDNA harboring plasmid. Serum samples were collected from lateral tail vain at time points indicated in the Figures. To determine the antigen-specific IgG titer in the serum of the individual mouse, ELISA were performed using 0.1 μg/ml antigen-coated assay plates (Maxisorp, Nunc 44-2404-21) following the standard procedure.

Example 2 [Yoshida et al., Vaccine 28: 3688 (2010)]

Construction of X:TNF-family extracellular domain polypeptide and Y: TNF-family extracellular domain polypeptide fusion protein.

Construction of CD40L-FasL-IgFc Plasmid DNA

To prepare the CD40⁺ B cell-specific CD40L-FasL-IgFc fusion protein mitogen gene construct, cDNAs encoding the extracellular domain of human CD40 ligand, extracellular domain of human Fas ligand, signaling sequence of oncostain M and CH2-CH3 domain of human IgG1 was generated by reverse transcriptase polymerase chain reaction (RT-PCR).

The extracellular domain of human CD40L [NM_000074, Homo sapiens CD40 ligand (CD40LG)] (amino acid residues 46-261) was amplified from human thymic RNA (Clontech B-D, Palo Alto, Calif., USA) using the following primers: forward 5′-CTTCATAGAAGGTTGGACAAGATA-3′ (SEQ ID NO: 43) and reverse 5′-GAGTTTGAGTAAGCC AAAGGACGT-3′ (SEQ ID NO: 44). The registered human CD40L message sequence is shown in FIG. 2 (SEQ ID NO: 1).

The signal peptide of Oncostatin M [NM_020530, Homo sapiens oncostatin M (OSM)] (amino acid residues 1-25) was amplified using the following primers: forward 5′-ATGGGGGTACTGCTCACACAGAGG-3′ (SEQ ID NO: 45) and reverse 5′-CATGCTCGCCATGCTTG GAAACAG-3′ (SEQ ID NO: 46). The registered human Oncostatin M message sequence is shown in FIG. 3 (SEQ ID NO: 3). These PCR products were subsequently utilized in a second round of PCR. Primers encoding the 3′ sequence of the Oncostatin M fragment and the 5′ portion of human CD40L were designed to overlap; annealing of the PCR products yielded a hybrid template. The template encoding the chimeric construct was selectively amplified using external primers specific for the 5′ region of Oncostatin M and the 3′ region of human CD40L. Each primer containing appropriate restriction sites (Nhe I/Xho I) for subcloning into the mammalian expression vector PCIneo (Promega, Madison, Wis.). The resulting PCR fragment was ligated into the PCIneo vector (OncoM-hCD40L/PCIneo), and transfected into E. coli competent cells, which were selected on an ampicillin-containing agar plate.

The hinge region, consisting of CH2 and CH3 domains (amino acid residues 219-447) of human IgG1, was amplified by RT-PCR from total human peripheral lymphocytes RNA, using the following primers: forward 5′-AAACTCGAGAAATCT TGTGACAAAACTCACACATGCCCACCGTG-3′ (SEQ ID NO: 47) and reverse 5′-CGTCTAGATCATTTACCCGGAGACAGGG AGAG-3′ (SEQ ID NO: 48), introducing the Xho I and Xba I sites, respectively (sites are underlined). The registered human IgG1 message sequence is shown in FIG. 4 (SEQ ID NO: 5). This PCR product was cloned into OncoM-hCD40L/PCIneo after digestion and purification (the resultant product coded as OncoM-CD40L-IgFc/PCIneo). The cDNA sequence of OncoM-CD40L-IgFc is shown in FIG. 5 (SEQ ID NO: 7). This fusion protein was a ˜50 kDa molecular mass in reduced SDS-PAGE and was designated as CD40L-IgFc in the text.

Chimeric Ig molecules expressing the extracellular portion of the human FasL gene and the human IgG1 constant domains were created as follows: the extracellular domain of human FasL (amino acid residues 108-281) was amplified by RT-PCR from total RNA of human thymus, using the following primers: forward 5′-CCGCTCGAGCAGCTCTTCC ACCTACAG-3′ (SEQ ID NO: 49) and reverse 5′-GGCCTCGAGCTTATATAAGCCGAAAAACGTC-3′ (SEQ ID NO: 50), including the Xho I sites, respectively (sites are underlined). The registered human FasL message sequence is shown in FIG. 6 (SEQ ID NO: 9). External primers encoding the 5′ portion and the 3′ portion of Oncostatin M, FasL and IgG1 were used to amplify the OncoM-hCD40L-hFasL-IgFc/PCIneo. Each primer contained appropriate restriction sites for subcloning into the PCIneo vector, yielding OncoM-hFasL-IgFc/PCIneo. This fusion protein was a ˜40 kDa molecular mass in reduced SDS-PAGE and was designated as FasL-IgFc in text. The cDNA sequence of OncoM-FasL-IgFc is shown in FIG. 7 (SEQ ID NO:

The extracellular domain of human FasL (amino acid residues 108-281) was cloned in frame at the 3′ end of the hCD40L in the OncoM-hCD40L-IgFc/PCIneo (the resultant product coded as CD40L-FasL-IgFc/PCIneo). This fusion protein was detected as a ˜70 kDa molecular mass in reduced SDS-PAGE and was designated as CD40L-FasL-IgFc in the text. All constructs were confirmed by DNA sequencing. The cDNA sequence of OncoM-CD40L-FasL-IgFc is shown in FIG. 8 (SEQ ID NO: 13). Plasmid DNA was purified by cesium chloride gradient ultracentrifugation.

Expression of CD40L-FasL-IgFc in Mammalian Cell Lines and Protein Purification from the Cell Culture Supernatants of Transfected Cells.

The pCI-neo harboring CD40L-FasL-IgFc cDNA construct, CD40L-FasL-IgFc/PCIneo was transfected in Chinese hamster ovarian fibroblastic cell (CHO cell) by electroporation technique. CHO cells (ATCC, CRL-9606) were maintained in Dulbecco's modified MEM (Invitrogen) supplemented with 1× Penicillin and Streptomycin (100× stock solution, Invitrogen), 1×L-glutamine (100× stock solution, Invitrogen) and 5% fetal bovine serum (FBS, Invitrogen). CHO cells were maintained in Culture flasks (Falcon 175 cm²) in 5% CO2 at 37° C. Cells were passaged weekly by 1/10 into a new flask. To split CHO cells to a new flask, medium was discarded from flask and replaced 3 ml 0.25% trypsin-EDTA medium (Invitrogen). After a 5 minute incubation at 37° C., cells were agitated by 5 ml pipette (Becton-Dickinson, Falcon) and harvested as a single cell suspension. Cells were pelleted by centrifuge (Sorvall RT 6000 Series tabletop centrifuge, 900 rpm for 6 minutes), resuspended to lml of DMEM, and then 0.1 ml of cell suspension was seeded to new flask with 20 ml of culture medium.

To transfect CD40L-FasL-IgFc/PCIneo expression vector to CHO cells, CHO cells were harvested by trypsin on day 4 after the split, washed once, then resuspended at a density of 10⁷ cells/ml in Na-phosphate buffered 0.9% NaCl solution. Expression plasmid (5 μg) was added to 0.5 ml of cell suspension in electroporation cuvette (Bio-Rad, 165-2081). The cuvette was chilled on ice 5 minutes prior to electroporation with a Gene-Pulser (Bio-Rad) at 250V, 975 μF. The cuvette was chilled again on ice for 30 minutes. The cells were washed once, resuspended with 10 mL DMEM tissue culture medium containing 5% FBS and then distributed to a 96 well flat bottom tissue culture plate at 100 μl/well. Twenty-four hours later, 100 μl of tissue culture medium containing 1 mg/ml of G418-sulfate (Geneticin, Invitrogen) was added to each well to select G418 resistant (positively transfected) cells. In 10 days, G418-resistant cells formed colonies detectable by microscopy. Supernatants were screened for the presence of the fusion protein by human immunoglobulin G Fc-specific ELISA, which detects the secreted fusion proteins. Cells expressing the fusion proteins were expanded in culture and harvested spent medium for protein purification. To obtain cell culture spent medium of the CD40L-FasL-IgFc CHO transfectant cell, cells were maintained in flasks (Falcon 300 cm²) with 300 ml of DMEM supplemented with 5% FBS.

Fusion proteins secreted in the spent medium of the CHO transfected cells were purified by Protein G column (HiTrap™ Protein G HP Columns GE Healthcare). Protein G columns (1 ml bed volume) were loaded with 500 ml of spent medium at flow rate 0.5 ml/min using chromatography pump (P500, Pharmacia) at 4° C. The column was washed with 20 ml of phosphate buffered saline (PBS) then, CD40L-FasL-IgFc was eluted with 2 ml of pH 2.7, 0.2 M Glycine-HCl. The eluate was neutralized with 0.2 ml of pH 9.0, 1.0 M potassium phosphate buffer, then dialyzed against 0.01M Sodium phosphate buffer (pH 7.2) for two days before filtered with 0.22 μm Millipore syringe filter (EW-29950-30) to make sterile fusion protein stock suitable for downstream cellular and biochemical analysis. The protein concentrations were measured using Pierce BCA protein assay kit (Thermo Scientific, 23225).

Purified CD40L-FasL-IgFc was first analysed by the Western blotting analysis.

Two μg of protein was subjected to SDS-PAGE under reduced conditions and probed by human IgG specific Western blotting. Twelve well precast gels, 10% Nu-PAGE (Invitrogen) were used with a MOPS-based running buffer. Thirteen pi of protein solution, 5 μl of loading buffer and 2 μl of 0.5 M DTT were mixed in 1.5 ml micro centrifuge tube, heated at 75° C. for 10 minutes before loading to each well of the Nu-PAGE Gel. The reduced protein samples were resolved for 50 minutes at 200 V in 1×MOPS running buffer (Invitrogen). Separated proteins were then transferred to PVDF membranes using transfer buffer containing 10% Methanol (Sigma). The PVDF membranes were blocked with 3% fat free milk in 0.1% Tween 20 (Sigma)-PBS for an hour at room temperature, then incubated with goat anti-human IgG HRP or rabbit anti-human FasL over night. The membrane was washed with 0.1% Tween 20 PBS and developed using chemiluminescence substrate [ECL detection buffer (Pierce)] and exposed to X-ray film (Kodak).

A dominant band at ˜75 kDa was observed and determined to be glycosylated CD40L-FasL-IgFc (FIG. 9A). The predicted molecular weight of the CD40L-FasL-IgFc is 70.6 kDa without glycosylation. The same protein sample was also subjected to SDS-PAGE under reduced conditions and probed by human FasL specific Western blotting. In paralleled experiment to FIG. 9A, the PVDF membrane was probed with rabbit anti-human FasL antibody overnight, washed with 0.1% Tween 20-PBS and then probed with anti-Rabbit IgG-HRP for 1 hour at room temperature. The results obtained from chemiluminescence and exposure of the membrane to X-ray film showed a similar picture of the protein bands as observed in the human IgG-specific Western blotting (FIG. 9B). The data indicate that the 70 kDa protein is most likely the protein band representing the monomer of the fusion protein CD40L-FasL-IgFc.

FIG. 9. Western Blotting Analysis of CD40L-FasL-IgFc.

CD40L-FasL-IgFc was subjected to SDS-PAGE under reduced conditions, probed by human IgG specific (A) or human FasL specific (B) western blotting. The dominant band at ˜75 kDa and corresponds to glycosylated CD40L-FasL-IgFc. The control is the fraction derived from the protein G column and contains non-specifically proteins (confirmed by Coomassie staining).

Study of Fas Binding by CD40L-FasL-IgFc.

Previous studies have not tested Fas-containing fusion protein constructs in which Fas ligand is located in the middle of two different polypeptide domains like CD40L-FasL-IgFc. Thus, it is important to test if CD40L-FasL-IgFc can bind to the cell surface Fas antigen. We tested the binding of CD40L-FasL-IgFc to the CD40 negative/Fas positive mouse T cell line EL4 (ATCC# TIB39). EL4 cells (5×10⁶) were incubated with 2 μg of CD40L-FasL-IgFc in 0.5 ml of DMEM on ice for 1 hour. Cells were washed with PBS and lysed by 0.5 ml of ice cold 1% NP-40 lysis buffer (50 mM Tris-buffer pH7.4, 250 mM NaCl, 5 mM EDTA, 0.1% NP-40, plus protease inhibitors). After removing the insoluble cell debris by micro centrifugation (11500 rpm for 1 hour at 4° C.), CD40L-FasL-IgFc was precipitated by Protein A/G agarose beads (Santa Cruz # sc-2003) overnight. The beads were washed with PBS, resuspended with 40 μl loading buffer containing DTT, heated for 10 minutes at 75° C. and then resolved by 10% Nu-PAGE with MOPS running buffer. PVDF membrane-transferred protein blots were probed with a mouse Fas-specific rat monoclonal antibody (Jo2 Becton Dickinson #554254), washed and subsequently probed with anti-mouse IgG-HRP detection antibody (Promega).

FIG. 10. Study of Fas Binding by CD40L-FasL-IgFc.

For a positive control (in the left-end lane), Fas molecule of EL4 cell lysate was immunoprecipitated with mouse Fas-specific rat monoclonal antibody (Jo2). For another control (in the middle lane) a fusion protein, CD40L-IgFc, was used instead of CD40L-FasL-IgFc for the incubation with EL4 before cell lysate preparation.

Analysis of the Function of CD40L-FasL-IgFc In Vitro.

CD40L-FasL-IgFc Fusion Protein Activates B Cells and Macrophages.

We found that following treatment with our fusion protein, the target human B lymphoma cells (Raji ATCC, CCL 86 and Daudi ATCC, CCL213) did not undergo cell death, while an antibody against human Fas (14G5) effectively killed B lymphomas in our study. In light of this result, we wished to examine the precise function associated with this fusion protein. We cultured human PBMCs and mouse spleen cells in the presence of CD40L-FasL-IgFc. Whole blood, obtained from a healthy donor, was twice diluted with Na-PBS (pH 7.2) and peripheral blood mononuclear cells (PBMCs) were separated by Ficoll density gradients (Ficoll-Paque Plus, Amarsham Biotech) by centrifugation (Sorvall RT 6000, 1500 rpm for 15 minutes at room temperature). The peripheral blood mononuclear cells were collected from the interphase between the PBS top layer and Ficoll plus bottom layer, washed with PBS and subjected to cell proliferation assays. After measuring the concentration of the cells using a hemocytometer, the cells were diluted in proliferation assay medium (RPMI1640, 5×10⁻⁵ M 2-mercaptoethanol, 1×L-glutamine, supplemented with 10% FBS). Human lymphocytes were stimulated for 3 days in 96 U bottom plates with various concentrations of recombinant fusion proteins. A ³H-Thymidine label was used to measure the uptake by the proliferating cells. Surprisingly, the cultures exhibited significant cell activation when viewed under a microscope, even at low concentration of fusion protein (0.1-0.01 μg/ml) (FIG. 11).

FIG. 11. Proliferation of Human PBMC Induced with CD40L-FasL-IgFc.

PBMCs were stimulated with various fusion proteins for three days prior to proliferation assay. Data indicate the means and standard deviations of triplicate samples. The data represent the five experiments with similar results. ▪: CD40L-IgFc, ●: FasL-IgFc, □: CD40L-IgFc+FasL-IgFc, ♦: IgFc, ◯: CD40L-FasL-IgFc.

In these experiments, the control protein used (FasL-IgFc) did not induce detectable cytotoxic activity in either the Raji human B cell line or the Fas apoptosis-sensitive Jurkat human T cell line. Culturing with FasL-IgFc also resulted in a small but increase in human PBL primary cultured cells. It was evident that our design of FasL fusion protein failed to create Fas apoptosis-inducing ligands. Another control protein, the CD40L-IgFc fusion protein, induced a mitogenic response. When the CD40L-IgFc and FasL-IgFc proteins were added as a mixture to PBMC cultures an additive effect of proliferation was observed. Overall, the CD40L-FasL-IgFc fusion protein greatly exceeded the mitogenic response induced by the co-stimulation with CD40L-IgFc and FasL-IgFc, and indeed the cell activation by CD40L-FasL-IgFc was specific for the non-T cell subsets, which contain CD40⁺ cells (FIG. 12). Additionally, in the study of the phenotype of CD40L-FasL-IgFc activated cells, we observed the activation in B cell (CD19⁺), and monocyte (CD14⁺) fractions increased by 30% and 15%, respectively in cultures stimulated for 5 days with CD40L-FasL-IgFc. The results suggest a novel activation mechanism of B-cell/monocyte induced by the co-clustering Fas and CD40 by CD40L-FasL-IgFc.

FIG. 12. Non-T Cell-Specific Stimulation by CD40L-FasL-IgFc.

PBL-T cells and PBL-non-T cells were stimulated with CD40L-FasL-IgFc (0.1 μM) for three days prior to proliferation assay. Data indicate the means and standard deviations of triplicate samples. The data represent the three experiments with similar results. C: control response, S: response against CD40L-FasL-IgFc.

This effect was not due to inadvertent contamination with endotoxin, as adsorption with Polymixin B did not attenuate the mitogenic activity of this fusion protein (FIG. 13).

FIG. 13. Effects of Polymixin B Pre-Absorption on the Stimulation by CD40L-FasL-IgFc.

CD40L-FasL-IgFc was pre-absorbed with Polymixin B-coated beads (Sigma). PBMCs were stimulated with this Polymixin-B pre-absorbed or unabsorbed CD40L-FasL-IgFc for three days prior to proliferation assays. Filled columns show responses against absorbed CD40L-FasL-IgFc. Open columns show responses against CD40L-FasL-IgFc prior to absorption. Data indicate the means and standard deviations of triplicate samples.

The CD40L-FasL fusion protein activates the NF-κB pathway and MEK1/2 ERK pathway.

Besides activating Caspase-dependent apoptotic pathways, Fas stimulates the NF-κB and ERK pathways. CD40 is also linked to stimulation of similar signaling pathways. Therefore, we hypothesized that the synergistic effect of Fas/CD40 activation is due to a convergence of signals at the level of the NF-κB and MEK1/2 ERK pathways. To test this, we assessed the effects of inhibitors specific for NF-κB or MEK1/2 on CD40L-FasL-IgFc-induced stimulation of PBL cells. We found that both inhibitors partially suppressed CD40L-FasL-IgFc-induced cell proliferation. Therefore the results pointed to a mechanism of activation by our fusion protein that involves both NF-κB and MEK1/2 activation (FIG. 14).

FIG. 14. Reduction of CD40L-FasL-IgFc-Induced Activation by Signaling Inhibitors.

PBMCs were stimulated with CD40L-FasL-IgFc for three days in the presence of NF-κB inhibitor (SN50) and MEK1/2 inhibitor (PD98059). Data indicate the means and standard deviations of triplicate samples. The data represent the three experiments with similar results.

CD40L-FasL Fusion Protein Induces B-Cell Differentiation In Vitro.

We next assessed the effect on IgG secretion by fusion protein-induced co-clustering of CD40 and Fas in B cells. PBL-B cells were incubated with CD40L-FasL-IgFc for four days and the supernatants were assayed for IgG- and IgM-specific trinitrophenyl (TNP) hapten [Galanaud et al., Cell. Immunol., 106:234 (1987)]. We found anti-TNP IgG was significantly increased in CD40L-FasL-IgFc-stimulated cultures, whereas cultures with either CD40L-IgFc or FasL-IgFc alone did not show significant changes in IgG secretion (FIG. 15).

FIG. 15. Induction of IgG Secretion in PBL Cells Stimulated with CD40L-FasL-IgFc.

PBMCs were stimulated with fusion proteins for four days. The relative concentrations of TNP-specific human IgG and IgM in supernatants were determined by ELISA assays. Data indicate the means and standard deviations of triplicate samples. The data represent the three experiments with similar results.

Moreover, we found an increased expression of PRDI-BF1 RNA, which indicates terminal B cell differentiation [Mock et al., Genomics, 37:24 (1996)] in cultures stimulated with CD40L-FasL-IgFc (FIG. 16). Taken together, the data suggests that co-clustering of CD40 and Fas by CD40L-FasL-IgFc induces B-cell differentiation in vitro.

FIG. 16. The Expression of PRDI-BF1 in Response to CD40L-FasL-IgFc Stimulation.

PBMCs were stimulated with fusion proteins (0.1 μM) for three days. The total RNA was assayed for the message of PRDI-BF1 by RT-PCR. Each sample was electrophoresed on agarose gels and stained with ethidium bromide (EtBr) to detect amplified fragments. The data represent the results from three experiments with similar results.

Study for the Function of CD40L-FasL-IgFc In Vivo.

The CD40L-FasL fusion protein boosts IgG response in vivo against antigens administrated simultaneously.

Stimulation of CD40⁺ cells by the CD40L-FasL-IgFc could allow this molecule to function as an adjuvant in vivo, by enhancing the activity of B cells and APCs. Many ligands specific for TLR family receptors act as adjuvants in experimental animals. These adjuvants augment the antibody and T cell responses against the co-injected vaccines such as type A influenza virus antigens. To determine if our fusion protein can behave as an adjuvant, we tested CD40L-FasL-IgFc in mice to see if it increases the immune response against co-injected protein antigens.

Anti-OVA Albumin (OVA) Immune Response.

Following immunization with a small dose of OVA (0.2 μs) by itself, a significant primary antibody production was not observed (FIG. 17). In contrast, the immunizations with a mixture of CD40L-FasL-IgFc and the OVA, resulted in the mice producing high titers of OVA-specific IgG antibodies, and this response was increased markedly following a boost by 0.2 μg OVA.

FIG. 17. Adjuvant Activity of CD40L-FasL-IgFc in Mice Against OVA.

Balb/c mice were immunized i.p. with 0.2 μg of OVA and 5 μg of CD40L-FasL-IgFc (indicated as Primed). On Day 14, blood samples were harvested and subsequently mice were injected with 0.2 μg of OVA (indicated as Boosted). Sera from day 14 and day 21 were analysed by ELISA for the IgG activity against OVA. The titer was calculated based on the dilutions and ELISA reader readings that first showed larger than twice of pre-immune sera readings at the same dilutions. Squares indicate mice immunized with OVA alone. Circles indicate mice immunized with both OVA and CD40L-FasL-IgFc. Numbers in squares and circles indicate individual mouse. The anti-HA activity of pre-immune serum from each mouse was equally low at less than 50 in titer.

Anti-Influenza Vaccine Response.

In the study similar to the OVA immunizations, in vivo adjuvant activity of CD40L-FasL-IgFc was investigated in mice using a specific human influenza vaccine as an antigen (FIG. 18). The vaccine, GSK VAXIGRIP, contains HA antigens of H1N1, H3N2 but the preparation is adjuvant-free. The vaccine dose (3 μg/mouse) used to prime was sufficient to induce a primary response detectable by vaccine-specific anti-IgG ELISA. The addition of CD40L-FasL-IgFc significantly increased the antibody response detected by ELISAs. Secondary responses following boosts were also far greater in groups primed with CD40L-FasL-IgFc than in the mice primed with vaccine only. Therefore this study demonstrated that the influenza HA antigen-specific immune response was augmented by CD40L-FasL-IgFc.

FIG. 18. Adjuvant Activity of CD40L-FasL-IgFc in Mice Against Influenza Hemaglutinin.

Balb/c mice were immunized i.p. with 3 μg of HA and 5 μg of CD40L-FasL-IgFc (indicated as Primed). On Day 14, blood samples were harvested from tail vain and subsequently mice were injected with 3 μg of HA (indicated as Boosted). Sera from day 14 and day 24 (for the study of secondary response) were analysed by ELISA for the IgG activity against HA. The titer was calculated based on the dilutions and ELISA reader readings that first showed larger than twice of pre-immune sera readings at the same dilutions. Squares indicate mice immunized with HA alone. Circles indicate mice immunized with HA and CD40L-FasL-IgFc. Numbers in squares and circles indicate individual mouse. The anti-HA activity of pre-immune serum from each mouse was equally low at less than 50 in titer.

The plasmid DNA of CD40L-FasL-IgFc increases anti-Influenza HA immune response in vivo.

Recently, attempts have been made to elicit pathogen-specific antibody and T cell responses using Plasmid DNAs. These Plasmid DNAs encode pathogen antigens that are to be used as vaccine reagents. In carrying out these experiments, immunizations were often performed in combination with adjuvants such as Alum or TLR ligands (i.e. CpG) [Daubenberger Curr Opin Mol Ther. 9:45(2007)]. Since we have demonstrated that our CD40L-FasL-IgFc possesses potent adjuvant effects against co-administrated protein antigens, we wished to test if the plasmid encoding the CD40L-FasL-IgFc would function as a DNA-adjuvant. Indeed, we observed an increased influenza vaccine-specific IgG response following the administration of the pCI-neo-CD40L-FasL-IgFc plasmid (CeCl2-purified) in conjunction with influenza vaccine (FIG. 19).

FIG. 19. Adjuvant Activity of Plasmid DNA of CD40L-FasL-IgFc in Mice Against Influenza Hemaglutinin.

Balb/c mice were immunized i.p. with 3 μg of HA and 3 μg of CD40L-FasL-IgFc/PCIneo (indicated as Primed). On Day 14, blood samples were harvested from tail vain and subsequently mice were injected with 3 μg of HA (indicated as Boosted). Sera from day 14 and day 24 (for the study of secondary response) were analysed by ELISA for the IgG activity against HA. The titer was calculated as descried in FIG. 18. Squares indicate mice immunized with HA alone. Circles indicate mice immunized with HA with plasmid. Numbers in squares and circles indicate individual mouse. The anti-HA activity of pre-immune serum from each mouse was equally low at less than 20 in titer.

Adjuvant activity of CD40L-FasL-IgFc on T cell responses against Influenza hemaglutinin.

Both the helper T cell response and the cytotoxic CD8⁺ T cell response are vital for the vaccine antigen-specific immune responses. Therefore this study was aimed at investigating the regulation of the HA-specific T cell response following CD40L-FasL-IgFc injection. Balb/c mice were immunized i.p. with 3 μg of HA and 3 μg of CD40L-FasL-IgFc. On Day 14, mice were injected with 3 μg of HA. Spleen cells harvested on day 60 (for the study of memory response) were analysed by proliferation assays against HA. Under sterile conditions, harvested spleens were minced in between a pair of slide glasses and red blood cells were removed by Gey's solution-treatment for 1 minute on ice. The prepared spleen cells (10⁶/ml) were stimulated in 96 well U bottom plates (10⁵/well) with 0.1 μg/ml HA for 48 hours in RPMI1640, 1×L-Glutamine (Invitrogen), 5×10⁻⁵ M 2-mercaptoethanol (Sigma) and 10% FBS (Invitrogen). The results showed the percent viabilities of cells measured by MTT assay performed by standard procedures. To measure the activity of CD8⁺ cytotoxic T cell response, monoclonal anti-L3T4 (anti-CD4) antibody (GK1.5, Pharmingen, 553726, 1 μg/ml) and monoclonal anti-class II MHC antibody (anti-I-E 14-4-4, Pharmingen, 558734, 1 μg/ml) were added in the culture medium. The presence of these antibodies blocks the CD4 T cell response as reported [Lim et al., Infect Immun., 63:4818 (1995)].

Interestingly, the addition of CD40L-FasL-IgFc lowered the HA-specific response by total T cells compared with HA only immunized spleen cells. In contrast to the response by total T cells, the CD8⁺ T cell response was only detectable in spleen cells immunized with CD40L-FasL-IgFc and not in mouse cells immunized with HA alone. The data showed strong adjuvant activity by CD40L-FasL-IgFc to antigen-specific CD8⁺ T cell responses in vivo.

FIG. 20. Adjuvant Activity of CD40L-FasL-IgFc in Mice Against Influenza Hemaglutinin-Specific T Cell Response.

A indicates total T cell response; B indicates CD8⁺ T cell response.

Percent viability is calculated based on the comparison with the non-stimulated cells (100%). The data shows the average and standard deviation derived from the triplicate sample. Not immunized: normal mice, Immunized: mice were immunized with HA only.

Example 3

Construction of a Fusion Protein Comprised of X: Immunoglobulin-Family Receptor Ligand Extracellular Domain Polypeptide, Y: TNF-Family Extracellular Domain Polypeptide Fusion Protein.

The B7-1(CD80)/B7-2(CD86)-CD28 pathway is the best-characterized T-cell co-stimulatory pathway. B7-1 and B7-2 are cell surface immunoglobulin family proteins expressed on macrophages and B cells. The CD28 signaling induced by B7-1 and B7-2 does not induce cell proliferation or cytokine secretion. It does, however, promote greatly enhanced activation events when T cells are simultaneously stimulated with T cell receptors. To determine if Fas signaling incorporate with CD28 signaling to elicit T cell proliferation, we constructed a B7-2-FasL-IgFc fusion protein. The result from the study of B7-2-FasL-IgFc was expected to address whether the conjugation of an immunoglobulin family co-stimulatory receptor ligand with a TNFR family death receptor ligand in the fusion protein mitogen formula (“X”-“Y”-IgFc) results in a novel mitogen.

Construction of B7-2-FasL-IgFc Plasmid DNA.

Registered message and amino acid sequence for human B7-2 is shown in FIG. 21 (SEQ ID NO: 15).

The extracellular domain of human B7-2 (amino acid residues: 24-247) was amplified by RT-PCR, from total RNA derived from Raji cells, using the following primers: forward 5′-GGACTCGAGGCTCCTCTGAAGATTCAAGC-3′ (SEQ ID NO: 51) and reverse 5′-AATCTCGAGA GGAATGTGGTCTGGGGGAG-3′ (SEQ ID NO: 52). The primers introduced Xho I sites useful for DNA subcloning (underlined). The PCR product was cloned in frame at the 3′ end of the hFasL in the OncoM-hFasL-IgGFc/PCIneo (the resultant product coded as OncoM-hB7-2-hFasL-IgGFc/PCIneo). The cDNA sequence and predicted amino acid sequence for this fusion construct is shown in FIG. 22 (SEQ ID NO: 17).

Chimeric Ig molecules expressing the extracellular protein of the human B7-2 gene and human IgG1 constant domains were created as follows: external primers encoding the 5′ portion and the 3′ portion of Oncostatin M, B7-2 and IgG1 were used to amplify the OncoM-hB7-2-IgGFc/PCIneo. Each primer contained appropriate restriction sites for subcloning into the PCIneo vector, yielding OncoM-B7-2-IgGFc/PCIneo (cDNA sequence and predicted amino acid sequence for this fusion construct is shown in FIG. 23, (SEQ ID NO: 19). This fusion protein, designated as B7-2-IgFc, was a ˜40 kDa molecular mass when subjected to SDS-PAGE under reduced conditions.

The transfection of B7-2-FasL-IgFc/PCIneo into CHO cell and the establishment of the stable transfectant cells were performed as described in the Example 2.

The Western blotting analysis for the Protein G column-purified B7-2-FasL-IgFc fusion protein was performed as described in the Example 2.

This fusion protein designated as B7-2-FasL-IgFc was detected as a ˜75 kDa molecular mass when subjected to SDS-PAGE under reduced conditions (FIG. 24).

The data indicated that the 75 kDa protein corresponds to the protein band representing the monomer of the fusion protein B7-2-FasL-IgFc.

FIG. 24. Western Blotting Analysis of B7-2-FasL-IgFc.

B7-2-FasL-IgFc was subjected to SDS-PAGE under reduced conditions, probed by human IgG specific (A) or human FasL specific (B) western blotting. The dominant band (˜75 kDa) corresponds to glycosylated B7-2-FasL-IgFc. The predicted molecular weight of the B7-2-FasL-IgFc is 72.5 kDa without glycosylation. The control is the fraction derived from the protein G column and contains non-specific proteins (confirmed by Coomassie staining).

Study of Fas binding by B7-2-FasL-IgFc.

To test if B7-2-FasL-IgFc binds to the cell surface Fas antigen, the binding of B7-2-FasL-IgFc to Fas positive mouse T cell line EL4 was tested using a similar approach described in Example 2.

FIG. 25. Study of Fas Binding by B7-2-FasL-IgFc.

For positive control (in the left end lane), Fas molecule of EL4 cell lysate was immunoprecipitated. In another control (in the middle lane) a fusion protein, CD40L-IgFc was used instead of B7-2-FasL-IgFc for the incubation with EL4 prior to cell lysate preparation.

Study for the Function of B7-2-FasL-IgFc In Vitro.

B7-2-FasL-IgFc Fusion Protein Activated T Cells.

PBMCs derived from normal healthy human adults were separated from whole blood using Ficoll-Paque Plus. Peripheral blood lymphocyte (PBL)-T cells, PBL-B cells, CD4⁺ T cells, CD8⁺ T cells were purified by negative selection using RosetteSep (StemCell Tec. Vancouver, BC, Canada), while non-T cells were purified by negative selection using pan-T magnet beads (M-450, Dynal, Lake Success, N Y, USA). The purity of human PBL-T cells was greater than 97% as determined by CD3 staining and flow cytometry. The residual T cell in non-T cell subsets was observed to be less than 5%. CD4⁺ and CD8⁺ T cells were purified to greater than 90% positive for CD4 and CD8, respectively as determined by flow cytometry studies.

Strikingly, when tested in primarily cells, the B7-2-FasL-IgFc fusion protein induced a strong T cell-specific mitogenic response (FIG. 26).

FIG. 26. Activation of CD4⁺ and CD8⁺ T Cells by B7-1-FasL-IgFc.

Total T cells, CD4⁺ T cells and CD8⁺ T cells were stimulated with either B7-2-FasL-IgFc or control fusion proteins. CD4⁺ T cells and CD8⁺ T cells were stimulated with various fusion proteins. After 72 hours in culture, cell proliferation was assessed. Data indicates the average and standard deviation of quadruplicate samples. The data represent the three experiments with similar results.

●: B7-2-FasL-IgFc, ▾: phytohemagglutinin, ▴: B7-2-IgFc, v: FasL-IgFc, ⋄: OKT3 and Δ: B7-2-IgFc+FasL-IgFc. In top panel

indicates the response by B cells against B7-2-FasL-IgFc (5 μg/ml).

The magnitude of proliferation induced by B7-2-FasL-IgFc was comparable to phytohemagglutinin-induced T cell mitogenesis, while neither B7-2-IgFc nor FasL-IgFc showed observable mitogenic effects in T cells. The proliferation of both CD4⁺ and CD8⁺ T cells increased by stimulation with B7-2-FasL-IgFc, but CD8⁺ T cells required greater than a 20 fold dose to achieve similar levels of proliferation to CD4⁺ T cells. The stimulation of T cells resulted in the secretion of IL-2 (FIG. 27).

FIG. 27. B7-2-FasL-IgFc Stimulates IL-2 Production of T Cells.

T cells were stimulated by B7-2-FasL-IgFc (1 μg/ml) or phytohemagglutinin (5 μg/ml) for 3 days. The concentration of IL-2 in culture supernatants was measured by ELISA assays. The lower limit of detection was 30 pg/ml in IL-2 assay. The data represent the three experiments with similar results.

Moreover, five days of culture with B7-2-FasL-IgFc showed a significant increase in the T-bet transcription factor RNA, but not that of GATA3 in cells (FIG. 28).

FIG. 28. Induction of T-Bet by B7-2-FasL-IgFc Activation.

CD4⁺ T cells were stimulated by B7-2-FasL-IgFc (0.1 μM) for 5 days before total RNA was harvested and tested for increased mRNA expression of T-bet (for Th1 differentiation) and GATA-3 (for Th2 differentiation) by RT-PCR. Each sample was electrophoresed on agarose gels and stained with ethidium bromide (EtBr) to detect amplified fragments: 206 bp for T-bet and 131 bp for GATA3. The data represent the three experiments with similar results.

These results indicated that B7-2-FasL-IgFc stimulated differentiation of CD4⁺ T cells in vitro by skewing towards Th1 subsets [Szabo et al., Cell. 100:655 (2000)]. As was observed in CD40L-FasL-IgFc-induced stimulation of B cells, the inhibitors for NF-κB pathway and ERK pathway attenuated T cell activation by B7-2-FasL-IgFc (FIG. 29).

FIG. 29. Reduction of B7-2-FasL-IgFc-Induced Activation by Signaling Inhibitors.

PBMCs were stimulated with B7-2-FasL-IgFc (0.1 μM) for three days in the presence of SN50 and PD98059. Data indicate the means and standard deviations of triplicate samples. The data represent the three experiments with similar results.

Study for the Function of B7-2-FasL-IgFc In Vivo.

B7-2-FasL-IgFc fusion protein represses the IgG response in vivo against antigens administrated simultaneously.

In reasons analogous to CD40L-FasL-IgFc, the stimulation of CD28⁺ T cells by B7-2-FasL-IgFc fusion protein could indicate that this fusion protein could function as an adjuvant in vivo by enhancing the activity of T cells. To determine if the fusion protein behaves as an adjuvant, we tested B7-2-FasL-IgFc in mice to see if it increases the immune response against co-injected HA antigens.

Anti-Influenza Vaccine Response.

The potential in vivo adjuvant activity of B7-2-FasL-IgFc fusion protein was investigated in mice using a specific human influenza vaccine as an antigen (FIG. 30). The vaccine dose (3 μg/mouse) used to prime was sufficient to induce a primary response detectable by HA-specific anti-IgG ELISAs. To our surprise, the administration of B7-2-FasL-IgFc repressed the antibody response detected by ELISAs. Secondary responses following boosts were also far lower in groups primed with vaccine only. Therefore this study demonstrated that the influenza HA antigen-specific immune response was repressed by B7-2-FasL-IgFc fusion protein.

FIG. 30. Immunosuppressant Activity of B7-2-FasL-IgFc in Mice Against Influenza Hemaglutinin.

Balb/c mice were immunized i.p. with 3 μg of HA and 5 μg of B7-2-FasL-IgFc (indicated as Primed). On Day 14, blood samples were harvested from tail vain and mice were subsequently injected with 3 μg HA (indicated as Boosted). Sera from day 14 and day 24 (for the study of secondary response) were analysed by ELISA for the IgG activity against HA. The titer was calculated based on the dilutions and ELISA readings that first showed larger than twice of pre-immune sera readings at the same dilutions. Squares indicate mice immunized with HA alone. Circles indicate mice immunized with HA with B7-2-FasL-IgFc. Numbers in squares and circles indicate individual mouse. The anti-HA activity of pre-immune serum from each mouse was equally low at less than 50 in titer.

The plasmid DNA of B7-2-FasL-fusion protein represses anti-Influenza HA immune response in vivo.

As an extension from the fusion protein result shown in FIG. 30, we tested if the plasmid DNA encoding the B7-2-FasL-IgFc/PCIneo could function as DNA-suppressant to the immune response against co-administrated vaccine antigen. Indeed, it was observed that a heavily repressed influenza vaccine-specific IgG response occurred following the administration of the B7-2-FasL-IgFc/PCIneo plasmid in conjunction with influenza vaccine (FIG. 31).

FIG. 31. Suppressant Activity of Plasmid DNA of B7-2-FasL-IgFc in Mice Against Influenza Hemaglutinin.

Balb/c mice were immunized i.p. with 3 μg of HA and 3 μg of B7-2-FasL-IgFc plasmid (indicated as Primed). On Day 14, blood samples were harvested from tail vain and mice were subsequently injected with 3 μg of HA (indicated as Boosted). Sera from day 14 and day 24 (for the study of secondary response) were analysed by ELISA for the IgG activity against HA. The titer was calculated as described in Example 2. Squares indicate mice immunized with HA alone. Circles indicate mice immunized with HA with plasmid. Numbers in squares and circles indicate individual mouse. The anti-HA activity of pre-immune serum from each mouse was equally low at less than 20 in titer.

Suppressant activity of B7-2-FasL-IgFc on T cell responses against Influenza hemaglutinin.

The results from the study of the antibody response in HA antigen and B7-FasL-IgFc administrated mice could indicate that T cell response to HA antigen is also suppressed in the mice. The study of B7-2-FasL-IgFc, therefore, investigated how the Influenza hemaglutinin-specific T cell response is regulated following B7-2-FasL-IgFc injection. As we expected, the addition of B7-2-FasL-IgFc suppressed the HA-specific response by total T cells compared with spleen cells from mice immunized with HA alone (FIG. 32 A). The CD8⁺ T cell response was also undetectable in spleen cells from mice immunized with B7-2-FasL-IgFc (FIG. 32B). The data showed the strong immunosuppressant activity by B7-2-FasL-IgFc to antigen-specific T cell responses in vivo.

FIG. 32. Immunosuppressant Activity of B7-2-FasL-IgFc in Mice Against Influenza Hemaglutinin-Specific T Cell Response.

Balb/c mice were immunized i.p. with 3 μg of HA and 5 μg of B7-2-FasL-IgFc. On Day 14, mice were injected with an additional 3 μg HA. Spleen cells from day 60 were analysed by proliferation assay against HA. Spleen cells stimulated for 48 hours with HA and were analysed for viability by MTT assay as described in the Example 2. A indicates total T cell response; B indicates CD8⁺ T cell response.

Percent viability is calculated based on the comparison with the non-stimulated cells (100%). The data shows the average and standard deviation derived from the triplicate samples. Not immunized: normal mice, Immunized: mice were immunized with HA alone.

Repression of Staphyloccocal enterotoxin B (SEB) induced splenomegaly by plasmid DNA of B7-2-FasL-IgFc.

Staphylococcal enterotoxins (SEA, SEB, SEC etc.) are a group of bacterial toxins, which activate T cells upon combining with class II MEW antigens on B cells or antigen presenting cells [Kotzin et al., Adv Immunol., 54:99 (1993)]. Those toxins are called “superantigens” and are known to activate T cells in vivo. Resulting from the in vivo activation of T cells, the injection of a superantigen such as SEB into mice causes splenomegaly within 48 hours. To characterize the repressor effect against T cell by plasmid DNA of B7-2FasL-IgFc, Balb/c mice (6 weeks of age) were intraperitonealy injected with 20 μg of SEB and 3 μg of B7-2-FasL-IgFc/PCIneo. Two days later, spleens were harvested aseptically and the weight of the spleen was measured by analytical balance (Mettler). The data indicated in FIG. 33 showed a strong repression of SEB-induced splenomegaly by plasmid DNA of B7-2-FasL-IgFc.

FIG. 33. The Repression of SEB-Induced Splenomegaly by Co-Administrated Plasmid DNA of B7-2-FasL-IgFc.

Three mice were used to each group. The data indicates the spleen weight average of three and the standard deviation. The spleen weight* indicates the weight at milligram order.

The data strongly suggests that the B7-2-FasL-IgFc construct could be used as the DNA suppressant in vivo and showed a potential of this fusion protein to apply to the diseases where in the immune suppression is necessary, such as autoimmune diseases and transplant rejection reaction.

Example 4

Construction of a Fusion Protein Comprising X: TNF-Family Extracellular Domain Polypeptide and Y: TNF-Family Extracellular Domain Polypeptide Fusion Protein.

Both CD40L-FasL-IgFc in Example 2 and B7-2-FasL-IgFc in Example 3 employed the death receptor ligand (FasL) extracellular domain as the domain “Y”. To further our understanding about the combination of the “Y” domain and “X” domain, which render the fusion protein mitogenic to target cells, a new set of T cell co-stimulatory receptor ligands were chosen. In the new gene construct, “X” is OX40 ligand (OX40L) extracellular domain and “Y” is 4-1BB ligand (4-1BBL) extracellular domain. Both OX40L and 4-1BBL are the TNF family ligands for TNFR family T cell co-stimulatory receptor OX40 and 4-1BB respectively. OX40 signal has a critical role in the T cell maintenance of an immune response to generate an antigen-specific memory response due to its ability to promote survival. 4-1BB signal contributes to the clonal expansion, survival, and development of T cells. 4-1BB can also enhance T cell apoptosis induced by TCR/CD3 triggered activation, and regulate CD28 co-stimulation to promote IL-2/interferon-γ producing Th1 cell responses.

Construction of OX40L-4-1BBL-IgFc Plasmid DNA.

Registered messages and amino acid sequences for human OX40L and 4-1BBL is shown in FIG. 34 (SEQ ID NO: 21) and FIG. 35 (SEQ ID NO: 23) respectively.

The extracellular domain of human OX40L [HUMGP34M, Homo sapiens mRNA for glycoprotein 34, complete cds] (amino acid residues: 51-183) was amplified by RT-PCR from human monocyte cell line THP-1, using the following primers: forward 5′-GGACTCGAGCAGGTATCACATCGGTATCC-3′ (SEQ ID NO: 53) including the Xho I site and reverse 5′-GGAACGCGTAAGGACACAGAATTCACCAG-3′ (SEQ ID NO: 54), including the Mlu I sites, respectively (sites are underlined). The PCR product was cloned in frame at the Xho I-Mlu I site of PCIneo.

The extracellular domain of human 4-1BBL (NP 003802, Homo sapiens tumor necrosis factor (ligand) superfamily, member 9) (amino acid residues: 49-254) was first amplified by RT-PCR from human monocyte cell line THP-1, using the following primers: forward 5′-GGAACGCGTGCCTGCCCTGGCCGTGTCCG-3′ (SEQ ID NO: 55) inducing the Mlu I site and reverse 5′-AATCTCGAGTTCCGACCTCGGTGAAGGGA-3′ (SEQ ID NO: 56), including the Kpn I sites, respectively (sites are underlined). The PCR product was cloned in frame at the Mlu I-Kpn I site of PCIneo.

Xba I fragment of IgFc was inserted in frame into Xba I site of PCIneo.

hOncoM fragment with Nhe I/Xho I restriction sites was the same as used to generate OncoM-B7-2-IgFc and inserted into the Nhe I-Xho I restriction site of PCIneo.

OX40L-IgFc/PCIneo or 4-1BB-IgFc/PCIneo was prepared by removing 4-1BBL or OX40L from OncoM-OX40L-4-1BBL-IgFc/PCIneo (designated as OX40L-4-1BBL-IgFc/PCIneo described previously in the text.

The cDNA sequence for of the fusion constructs, OX40L-4-1BBL-IgFc/PCIneo, OX40L-IgFc/PCIneo and 4-1BB-IgFc/PCIneo are shown in FIG. 36 (SEQ ID NO: 25), 37 (SEQ ID NO: 27) and 38 (SEQ ID NO: 29) respectively.

The transfection of OX40L-4-1BBL-IgFc/PCIneo into CHO cell and the establishment of the stable transfectant cells were performed as described in the Example 2.

The Western blotting analysis for the Protein G column purified OX40L-4-1BBL-IgFc fusion protein was performed as described in the Example 2

This fusion protein designated as OX40L-4-1BBL-IgFc was detected as a ˜70 kDa molecular mass in reduced SDS-PAGE (FIG. 39). The data indicate that the 70 kDa protein corresponds to the protein band representing the monomer of the fusion protein OX40L-4-1BBL-IgFc.

FIG. 39. Western Blotting Analysis of OX40L-4-1BBL-IgFc.

The OX40L-4-1BBL-IgFc fusion protein was subjected to SDS-PAGE under reduced conditions and probed by human IgG specific (A) or human OX40 ligand specific (B) western blotting. The dominant band at ˜70 kDa corresponds to glycosylated OX40L-4-1BBL-IgFc protein. The presumed molecular weight of OX40L-4-1BBL-IgFc is 66.4 kDa without post-translational modification.

Study for the Function of OX40L-4-1BBL-IgFc In Vitro.

OX40L-4-1BBL-IgFc Fusion Protein Activated PBMCs.

Peripheral blood mononuclear cells from normal healthy human adults were separated from whole blood using Ficoll-Paque Plus. Unexpectedly, when tested in PBMCs, the mixture of OX40L-IgFc and 4-1BBL-IgFc induced a mitogenic response. The OX40L-4-1BBL-IgFc fusion protein, however, induced a far greater mitogenic response (FIG. 40).

FIG. 40. Activation of PBMCs by OX40L-4-1BBL-IgFc.

PBMCs were stimulated with OX40L-4-1BBL-IgFc (1 μg/ml) and control fusion proteins: OX40L-IgFc (1 μg/ml), 4-1BBL-IgFc (1 μg/ml) and OX40L-IgFc (0.5 μm/ml)+4-1BBL-IgFc (0.5 μm/ml). Cell proliferation was assessed following 72 hours in culture. Data indicates the average and standard deviation of quadruplicate samples.

The plasmid DNA of OX40L-4-1BBL-IgFc fusion protein increases anti-Influenza hemaglutinin immune response in vivo.

We next tested if the plasmid encoding the OX40L-4-1BBL-IgFc fusion protein would function as DNA-adjuvant. Significantly, we observed an increased influenza vaccine-specific IgG response following the administration of the OX40L-4-1BBL-IgFc/PCIneo plasmid in conjunction with influenza vaccine in mice (FIG. 41).

FIG. 41. Adjuvant Activity of OX40L-4-1BBL-IgFc/PCIneo in Mice Against Influenza Hemaglutinin.

Balb/c mice were immunized i.p. with 3 μg of HA and 3 μg of OX40L-4-1BBL-IgFc/PCIneo plasmid (indicated as Primed). On Day 14, blood samples were harvested and mice were subsequently injected with 3 μg of HA (indicated as Boosted). Sera from day 14 and day 24 were analysed by ELISA for the IgG activity against HA. The titer was calculated as described in Example 2. Squares indicate mice immunized with HA alone. Circles indicate mice immunized with HA with plasmid. Numbers in squares and circles indicate individual mouse. The anti-HA activity of pre-immune serum from each mouse was equally low at less than 20 in titer.

Poor activity of OX40L-4-1BBL-IgFc/PCIneo on T cell responses against Influenza hemaglutinin.

The study of OX40L-4-1BBL-IgFc was extended to investigate how the Influenza hemaglutinin-specific T cell response is regulated following OX40L-4-1BBL-IgFc/PCIneo injection (FIG. 42). Interestingly, the addition of OX40L-4-1BBL-IgFc/PCIneo repressed the HA-specific response by total T cells compared with spleens from mice immunized with HA alone. The response by total T cells and CD8⁺ T cell response was undetectable in spleen cells from mice immunized with HA and OX40L-4-1BBL-IgFc/PCIneo and it was nearly identical to cells from mice immunized with HA alone. The data showed poor adjuvant activity by OX40L-4-1BBL-IgFc/PCIneo to antigen-specific T cell responses in vivo, which contrasted to the strong adjuvant activity to antibody response by B cells.

FIG. 42. Adjuvant Activity of OX40L-4-1BBL-IgFc in Mice Against Influenza Hemaglutinin-Specific T Cell Response.

Balb/c mice were immunized intraperionealy with 3 μg of HA and 3 μg of OX40L-4-1BBL-IgFc/PCIneo plasmid. On Day 14, mice were injected with and additional 3 μg of HA. Spleen cells from day 60 were analysed by proliferation assay against HA. Spleen cells stimulated for 48 hours with HA and were analysed for viability by MTT assay as described in the Example 2. A indicates total T cell response; B indicates the CD8⁺ T cell response.

Percent viability is calculated based on the comparison with the non-stimulated cells (100%). The data shows the average and standard deviation derived from the triplicate samples. Not immunized: normal mice; Immunized: mice were immunized with HA only.

Example 5

Construction of a fusion protein comprising X: TNF-family ligand extracellular domain polypeptide and Y: immunoglobulin-family receptor extracellular domain polypeptide fusion protein.

In the additional combination of “X” and “Y” of the X—Y-IgFc fusion protein mitogens, “X” the extracellular domain of CD40L and “Y” the extracellular domain of ICOS were chosen. ICOS is an inducible co-stimulatory molecule on T cells and its expression depends on the activation state of T cells. ICOS, a member of CD28/CTLA4 family, is not categorized as the ligand but is a receptor of which the ligand (ICOS ligand) is expressed on B cells and antigen presenting cells. Like the CD40/CD40L interaction and many other receptor/cell surface-expressed contra-receptor (ligand) interaction, the interaction between ICOS and ICOS ligand is known to stimulate bi-directional signaling, which co-stimulates both T cell (by ICOS signaling) and B cell (by ICOS ligand signaling). Accordingly, the CD40L-ICOS-IgFc could stimulate both B cells and antigen presenting cells. The purpose of developing this fusion protein was to test if the extracellular domain of the immunoglobulin family co-stimulation receptor can be used as the domain “Y” of a fusion protein mitogen.

Construction of CD40L-ICOS-IgFc Plasmid DNA.

Registered RNA message and predicted amino acid sequence for human ICOS is shown in FIG. 43 (SEQ ID NO: 31).

The extracellular domain of human ICOS [NM_012092, Homo sapiens inducible T-cell co-stimulator (ICOS)] (amino acid residues: 20-141) was first amplified by RT-PCR from total RNA of PHA-stimulated human PBMCs, using the following primers: forward 5′-GGACTCGAGGGAGAAATCAATGGTTCTGC-3′ (SEQ ID NO: 57) and reverse 5′-AATCTCGAGG AACTTCAGCTGGCAACAAA-3′ (SEQ ID NO: 58), including the Xho I sites, respectively (sites are underlined). The PCR product was cloned in frame at the 3′ end of the hCD40L in the OncoMCD40L-IgGFc/PCIneo. The resultant product, OncoM-hCD40L-hICOS-IgGFc/PCIneo was designated as CD40L-ICOS-IgFc/PCIneo.

The cDNA sequence for this fusion construct is shown in FIG. 44 (SEQ ID NO: 33).

CHO cells were transfected with CD40L-ICOS-IgFc/PCIneo as described in Example 2. The CD40L-ICOSL-IgFc protein secreted in the culture supernatant of the stable CHO transfectant was purified by protein G column. Purified CD40L-ICOSL-IgFc was analysed by SDS-PAGE Western blotting specific for human IgG and human CD40 ligand (FIG. 45).

FIG. 45. Western Blotting Analysis of CD40L-ICOS-IgFc.

Purified CD40L-ICOS-IgFc was subjected to SDS-PAGE under reduced conditions, probed by human IgG specific (A) or human CD40L specific (B) western blotting. The dominant band at ˜70 kDa corresponds to glycosylated CD40L-ICOS-IgFc. The predicted molecular weight of the CD40L-ICOS-IgFc is 64.4 kDa without post-translational modification. The data indicate that the ˜70 kDa protein is the protein band representing the monomer of the fusion protein CD40L-ICOS-IgFc.

Study for the Function of CD40L-ICOS-IgFc/PCIneo In Vivo.

The plasmid DNA of CD40L-ICOS-IgFc-fusion protein increases anti-Influenza hemaglutinin immune response in vivo.

We tested if the plasmid DNA encoding the CD40L-ICOS-IgFc fusion protein would function as a DNA-adjuvant similar to CD40L-FasL-IgFc in Example 2. Indeed, increased influenza vaccine-specific IgG response was observed following the administration of the CD40L-ICOS-IgFc/PCIneo plasmid in conjunction with influenza vaccine (FIG. 46).

FIG. 46. Adjuvant Activity of Plasmid DNA of CD40L-ICOS-IgFc in Mice Against Influenza Hemaglutinin.

Balb/c mice were immunized i.p. with 3 μg of HA and 3 μg of CD40L-ICOS-IgFc plasmid. On Day 14, mice were injected with an additional 3 μg HA. Sera from day 14 and day 24 were analysed by ELISA for the IgG activity against HA. The titer was calculated as descried in Example 2. Squares indicate mice immunized with HA alone. Circles indicate mice immunized with HA with plasmid. Numbers in squares and circles indicate individual mouse. The anti-HA activity of pre-immune serum from each mouse was equally low at less than 20 in titer.

Effect of plasmid DNA of CD40L-ICOS-IgFc on T cell responses against Influenza hemaglutinin.

The study of CD40L-ICOS-IgFc was furthered by investigating how the Influenza hemaglutinin-specific T cell response is regulated following CD40L-ICOS-IgFc/PCIneo injection (FIG. 47). Interestingly, the addition of CD40L-ICOS-IgFc/PCIneo lowered the HA-specific response by total T cells compared with spleen cells derived from mice immunized with HA alone. In contrast to the response by total T cells, the CD8⁺ T cell response was only detectable in spleen cells from mice immunized with HA and CD40L-ICOS-IgFc/PCIneo whereas it was not detected in spleen cells from mice immunized with HA alone. The data showed the adjuvant activity by CD40L-ICOS-IgFc/PCIneo to antigen-specific CD8⁺ T cell responses in vivo. The activation of CD8⁺ T cells, however, was lower than that observed by CD40L-FasL-IgFc treated mice spleen cells.

FIG. 47. Adjuvant Activity of CD40L-ICOS-IgFc/PCIneo in Mice Against Influenza Hemaglutinin-Specific T Cell Response.

Balb/c mice were immunized i.p. with 3 μg of HA and 3 μg of CD40L-ICOS-IgFc/PCIneo plasmid. On Day 14, mice were injected with an additional 3 μg of HA. Spleen cells from day 60 were analysed by proliferation assay against HA. Spleen cells stimulated for 48 hours with HA and were analysed for viability by MTT assay as described in the Example 2. A indicates total T cell response; B indicates CD8⁺ T cell response. Percent viability is calculated based on the comparison with the non-stimulated cells (100%). The data shows the average and standard deviation derived from the triplicate samples. Not immunized: normal mice; Immunized: mice were immunized with HA only.

The results indicated that the extracellular domain of immunoglobulin family co-stimulation receptor, but not the ligand itself, is also usable to create a novel fusion protein mitogen.

Example 6

Construction of a fusion protein comprised of X: NGFβ-family cytokine, Y: TNF family extracellular domain polypeptide fusion protein.

In another variation of “X” and “Y” of the X—Y-IgFc fusion protein mitogens, “X” nerve growth factor (NGFβ) and “Y” the extracellular domain of FasL were chosen. NGFβ is a NGFβ family cytokine and an important factor for the survival, differentiation and maintenance of the sensory and sympathetic neurons. Like other members of NGFβ family cytokines, NGFβ possesses various functions, one of which is to promote B cell growth [Brodie and Gelfand, J Immunol. 148:3492 (1992)]. Accordingly, the NGFβ-FasL-IgFc could stimulate B cells and antigen presenting cells, which will promote the antibody response by B cells in vivo. This fusion protein will also tested to see whether the NGF-family cytokine can be used as the domain of a fusion protein mitogen.

Construction of NGFβ-FasL-IgFc Plasmid DNA.

Registered RNA message and amino acid sequence for human NGFβ is shown in FIG. 48 (SEQ ID NO: 35).

The human NGFβ (NM_002506, Homo sapiens nerve growth factor (beta polypeptide)) (amino acid residues: 122-241) was first amplified by RT-PCR from total RNA of PHA-stimulated human PBMCs, using the following primers: forward 5′-GGACTCGAGTCA TCATCCCATCCCATCTT-3′ (SEQ ID NO: 59) and reverse 5′-AATCTCGAG GGCTCTTCTCACAGCC TTCC-3′ (SEQ ID NO: 60), including the Xho I sites, respectively (sites are underlined). The PCR product was cloned in frame at the 5′ end of the hFasL in the OncoMFasL-IgGFc/PCIneo. The resultant product OncoM-hNGFβ-hFasL-IgGFc/PCIneo was designated as NGFβ-FasL-IgFc/PCIneo.

The cDNA sequence for this fusion construct is shown in FIG. 49 (SEQ ID NO: 37).

CHO cells were transfected with NGFβ-FasL-IgFc/PCIneo as described in Example 2. The NGFβ-FasL-IgFc protein secreted in the culture supernatant of the stable CHO transfectant was purified by protein G column. Purified NGFβ-FasL-IgFc was analysed by SDS-PAGE Western blotting specific for human IgG and human Fas ligand (FIG. 50).

FIG. 50. Western Blotting Analysis of NGFβ-FasL-IgFc.

Purified NGFβ-FasL-IgFc was subjected to SDS-PAGE under reduced conditions, and probed by human IgG specific (A) or human FasL specific (B) western blotting. The dominant band at ˜65 kDa corresponds to glycosylated NGFβ-FasL-IgFc protein. The predicted molecular weight of the NGFβ-FasL-IgFc is 60.5 kDa without post-translational modification. The data indicate that the ˜65 kDa protein the protein band representing the monomer of the fusion protein NGFβ-FasL-IgFc.

Study for the Function of NGFβ-FasL-IgFc/PCIneo In Vivo.

The plasmid DNA of the NGFβ-FasL-IgFc fusion protein increases anti-Influenza HA immune response in vivo.

We tested if the plasmid DNA encoding the NGFβ-FasL-IgFc fusion protein would function as DNA-adjuvant in a manner similar to the CD40L-FasL-IgFc fusion protein described in Example 2. Indeed, a largely increased influenza vaccine-specific IgG response was observed following the administration of the NGFβ-FasL-IgFc/PCIneo plasmid in conjunction with influenza vaccine (FIG. 51).

FIG. 51. Adjuvant Activity of Plasmid DNA of NGFβ-FasL-IgFc in Mice Against Influenza Hemaglutinin.

Balb/c mice were immunized i.p. with 3 μg of HA and 3 μg of NGFβ-FasL-IgFc plasmid (indicated as Primed). On Day 14, blood samples were harvested from tail vain and mice were injected with and additional 3 μg HA (indicated as Boosted). Sera from day 14 and day 24 were analysed by ELISA for the IgG activity against HA. The titer was calculated as described in Example 2. Squares indicate mice immunized with HA alone. Circles indicate mice immunized with HA with plasmid. Numbers in squares and circles indicate individual mouse. The anti-HA activity of pre-immune serum from each mouse was equally low at less than 20 in titer.

Effect of plasmid DNA of NGFβ-FasL-IgFc on T cell responses against Influenza hemaglutinin.

The study of NGFβ-FasL-IgFc investigated how the HA-specific T cell response is regulated following NGFβ-FasL-IgFc/PCIneo injection (FIG. 52). Similarly to results observed following CD40L-FasL-IgFc injection, the addition of NGFβ-FasL-IgFc/PCIneo lowered the HA-specific response by total T cells compared with spleen cells derived from mice immunized with HA alone. In contrast to the response by total T cells, CD8⁺ T cell response was increased to the level nearly equal to that observed by CD40L-FasL-IgFc-treated mouse cells and only detectable in spleen cells immunized HA and NGFβ-FasL-IgFc/PCIneo. The data showed significant adjuvant activity by NGFβ-FasL-IgFc/PCIneo to HA-specific CD8⁺ T cell responses in vivo.

FIG. 52. Adjuvant Activity of NGFβ-FasL-IgFc/PCIneo in Mice Against Influenza Hemaglutinin-Specific T Cell Response.

Balb/c mice were immunized i.p. with 3 μg of HA and 3 μg of NGFβ-FasL-IgFc/PCIneo plasmid. On Day 14, mice were injected with an additional 3 μg of HA. Spleen cells from day 60 were analysed by proliferation assay against HA. Spleen cells stimulated for 48 hours and were analysed for viability by MTT assay as described in the Example 2. A indicates total T cell response; B indicates CD8⁺ T cell response. Percent viability is calculated based on the comparison with the non-stimulated cells (100%). The data shows the average and standard deviation derived from the triplicate samples. Not immunized: normal mice, Immunized: mice were immunized with HA only.

Example 7

Construction of a X: interleukin 2-family cytokine, Y: TNF family extracellular domain polypeptide fusion protein.

In another variation of “X” and “Y” of the X-Y-IgFc fusion protein mitogens, “X” interleukin-2 (IL-2) and “Y” the extracellular domain of FasL were chosen. IL-2 is an IL-2 family cytokine and an important factor for the growth of T cells and the survival of T cells and B cells. Both T cells and B cells express receptors for IL-2 and it is possible both T and B cells would both be stimulated by the fusion protein IL-2-FasL-IgFc administration in vivo. This study also tested if the IL-2-family cytokines (IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21) can be used as the domain of a fusion protein mitogen.

Construction of IL-2-FasL-IgFc Plasmid DNA.

Registered RNA message and amino acid sequence for human IL-2 is shown in FIG. 53 (SEQ ID NO: 39).

The human IL-2 (NM_000586, Homo sapiens interleukin-2) (amino acid residues: 21-154) was amplified by RT-PCR from amplified by RT-PCR from total RNA of PHA-stimulated human PBMCs, using the following primers: forward 5′-GGAGAATTCGCA CCTACTTCAAGTTCTAC-3′ (SEQ ID NO: 61). and reverse 5′-AATACGCGTAGTCAGTGTTGAGAT GCTGCT-3′ (SEQ ID NO: 62), including the EcoRI and Mlu I sites, respectively (sites are underlined). The PCR product was cloned in frame at the 5′ end of the hFasL by replacing the EcoR I/Mlu I fragment of hB7-2 of OncoM-hB7-2-hFasL-IgGFc/PCIneo following the double digestion with EcoRI and Mlu I. The resultant product OncoM-hIL-2-hFasL-IgGFc/PCIneo was designated as IL-2-FasL-IgFc/PCIneo.

The cDNA sequence for this fusion construct is shown in FIG. 54 (SEQ ID NO: 41).

CHO cells were transfected with IL-2-FasL-IgFc/PCIneo as described in Example 2. The IL-2-FasL-IgFc/PCIneo protein secreted in the culture supernatant of the stable CHO transfectant was purified by protein G column. Purified IL-2-FasL-IgFc/PCIneo was analysed by SDS-PAGE Western blotting specific for human IgG and human Fas ligand (FIG. 55).

FIG. 55. Western Blotting Analysis of IL-2-FasL-IgFc.

Purified IL-2-FasL-IgFc was subjected to SDS-PAGE under reduced conditions and probed by human IgG specific (A) or human FasL specific (B) western blotting. The dominant band at ˜70 kDa corresponds to glycosylated IL-2-FasL-IgFc protein. The predicted molecular weight of IL-2-FasL-IgFc protein is 65.4 kDa without post-translational modification. The data indicate that the ˜70 kDa protein is the protein band representing the monomer of the fusion protein IL-2-FasL-IgFc.

Study for the Function of IL-2-FasL-IgFc/PCIneo In Vivo.

The plasmid DNA of the IL-2-FasL-IgFc fusion protein increases anti-Influenza hemaglutinin immune response in vivo.

We tested if the plasmid DNA encoding the IL-2-FasL-IgFc fusion protein would function as a DNA-adjuvant similar to that observed with the CD40L-FasL-IgFc fusion protein outlined in Example 2. Indeed, increased influenza vaccine-specific IgG responses were observed following the administration of the IL-2-FasL-IgFc/PCIneo plasmid in conjunction with influenza vaccine (FIG. 56).

FIG. 56. Adjuvant Activity of Plasmid DNA of IL-2-FasL-IgFc in Mice Against Influenza Hemaglutinin.

Balb/c mice were immunized i.p. with 3 μg of HA and 3 μg of IL-2-FasL-IgFc plasmid (indicated as Primed). On Day 14, blood samples were harvested from tail vain and mice were injected with an additional 3 μg HA (indicated as Boosted). Sera from day 14 and day 24 were analysed by ELISA for the IgG activity against HA. The titer was calculated as described in Example 2. Squares indicate mice immunized with HA alone. Circles indicate mice immunized with HA with plasmid. Numbers in squares and circles indicate individual mouse. The anti-HA activity of pre-immune serum from each mouse was equally low at less than 20 in titer.

Effect of plasmid DNA of IL-2-FasL-IgFc on T cell responses against Influenza hemaglutinin.

The inventor investigated how the Influenza hemaglutinin-specific T cell response is regulated following IL-2-FasL-IgFc/PCIneo injection in mice (FIG. 57). Interestingly, the addition of IL-2-FasL-IgFc/PCIneo repressed the HA-specific response by total T cells compared with spleen cells derived from mice immunized with HA alone. The CD8⁺ T cell response was not increased significantly but still a little higher than that observed by treatment of mice with B7-2-FasL-IgFc described earlier. The data showed that the adjuvant activity by IL-2-FasL-IgFc/PCIneo to antigen-specific T cell response in vivo is marginal.

FIG. 57. Suppressant Activity of IL-2-FasL-IgFc/PCIneo in Mice Against Influenza Hemaglutinin-Specific T Cell Response.

Balb/c mice were immunized i.p. with 3 μg of HA and 3 μg of IL-2-FasL-IgFc/PCIneo plasmid. On Day 14, mice were injected with an additional 3 μg of HA. Spleen cells from day 60 were analysed by proliferation assay against HA. Spleen cells stimulated for 48 hours with HA and were analysed for viability by MTT assay as described in the Example 2. A indicates total T cell response; B indicates CD8⁺ T cell response.

Percent viability is calculated based on the comparison with the non-stimulated cells (100%). The data shows the average and standard deviation derived from the triplicate samples. Not immunized: normal mice, Immunized: mice were immunized with HA only.

The Examples presented supra indicate that the current invention can generate a variety of fusion protein mitogens, which acquire different functions. Some of the fusion proteins possessed unique characteristics, which could be beneficial in the treatment of various human diseases. It is also anticipated that, based on these examples, the present invention will provide novel methods of treatment of diseases that either enhance or repress cellular and humoral immunity. Diseases or conditions that are viable targets for this mode of treatment include chronic and debilitating human diseases such as cancer and other proliferative diseases, infectious diseases, autoimmunity, allergic conditions, inflammatory conditions such as arteriosclerosis and organ transplant rejection. The invention broadly encompasses the use of the fusion proteins and plasmid DNAs for treatment or prevention of diseases wherein enhanced or reduced antigen specific cellular immunity is desirable.

It is to be understood that the invention is not limited to the embodiments listed above and the right is reserved to the illustrated embodiments and all modifications coming within the scope of the following claims.

The various references to journals, patents, and other publications which are cited herein comprise the state of the art and are incorporated by reference as though fully set forth. 

1. An immunostimulatory fusion polypeptide comprising in N-terminal to C-terminal direction a polypeptide comprising the receptor binding domain of nerve growth factor beta, a polypeptide comprising the receptor binding domain of Fas ligand, and a polypeptide comprising the human immunoglobulin Fc domain.
 2. A recombinant nucleic acid (cDNA) comprising a polynucleotide sequence that encodes the fusion polypeptide of claim
 1. 3. A cDNA expression vector containing a fusion polynucleotide sequence according to claim
 2. 4. A cDNA expression vector of claim 3, which is selected from a plasmid, recombinant virus, and episomal vector.
 5. A cell line that comprises the cDNA expression vector of claim
 3. 6. The immunostimulatory fusion polypeptide of claim 1, which is substantially pure. 